Regulation of fungal gene expression

ABSTRACT

Disclosed herein are methods for regulating fungal gene expression, and reagents for carrying out those methods.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of Ser. NO. 09/189,462 filed Nov. 10,1998 now U.S. Pat. No. 6,303,302, which claims benefit from provisionalapplications “Fungal Switching” (U.S. Ser. No. 60/066,129), “Candidaalbicans RIM1 Gene is Essential for Invasive Hyphal Growth” (U.S. Ser.No. 60/066,308), “Candida albicans RIM1 Gene is Essential for InvasiveHyphal Growth” (U.S. Ser. No. 60/066,462), “Novel Screens and SelectionsBased on Fungal Invasion: Tools for New Drug Discovery” (U.S. Ser. No.60/078,610), and “Fungal-Specific AFL1 Gene and its Function inAntifungal Drug Screening and in the Enhancement of Fungal ProductExpression” (U.S. Ser. No. 60/094,523), which were filed on Nov. 19,1997, Nov. 21, 1997, Nov. 24, 1997, Mar. 19, 1998, and Jul. 29, 1998,respectively.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

The invention was supported, in whole or in part, by funding from theGovernment. The Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

The present invention relates to methods of identifying and isolatinggenes which are involved in the regulation of fungal gene expression.The invention also relates to methods useful for identifying fungalvirulence factors. It further relates to a method of identifying agentswhich increase or decrease the expression or activity of a gene thatregulates or is required for fungal pathogenesis. The invention alsorelates to the use of such agents as fungicides or fungistats.

Fungi are a large and diverse group of organisms with enormousimportance to humans. Pathogenic fungi are a significant cause of humandisease, particularly in the rapidly increasing proportion of thepopulation whose immune system has been compromised by disease,chemotherapy, or immunosuppressive drugs. A wide variety ofplant-pathogenic fungi (e.g., blights, rusts, molds, smuts, mildews)cause huge food crop loss and damage to ornamental plants. Plantdiseases are caused by a myriad of invasive fungal pathogens fallinginto many genera, for example: soft rot (e.g., Rhizopus), leaf curl(e.g., Taphrina), powdery mildew (e.g., Sphaerotheca), leaf spots (e.g.,Fulvia), blight (e.g., Alternaria), blast (e.g., Magnaporthe), black rot(e.g., Guignardia), scab (e.g., Venturia), wilts (e.g., Fusarium), rusts(e.g., Puccinia), smuts (e.g., Ustilago), and cankers (e.g.,Rhizoctonia). In addition, fungal species are the commercial source of agreat many medicinally useful products, such as antibiotics (e.g.,beta-lactam antibiotics such as penicillin, cephalosporin, and theirderivatives), anti-hypercholesterolemic agents (e.g., lovastatin andcompactin), immunosuppressives (e.g., cyclosporin), and antifungal drugs(e.g., pneumocandin and echinocandin). All of these drugs are fungalsecondary metabolites, small secreted molecules that fungi utilizeagainst competitors in their microbial environment. Finally, fungi alsoproduce commercially important enzymes (e.g., cellulases, proteases, andlipases) as well as other products (e.g., citric acid, gibberellic acid,natural pigments, and flavorings).

The specifics by which fungi invade their growth substrate are notunderstood in detail. However, two important themes regarding the fungalinvasion process have emerged in recent years. First, important humanfungal pathogens, such as Candida sp., Aspergillus sp., Mucor sp.,Rhizopus sp., Fusarium sp, Penicillium marneffei, Microsporum sp. andTrichophyton sp. invade through host tissues as filamentous hyphae. Thevirulence of Candida (C.) albicans has been shown to be dependent uponinvasion of host tissues; mutations in any of several genes required forinvasive growth substantially reduce virulence in a mouse model ofsystemic infection. Pathogenesis of the plant fungal pathogen Ustilago(U.) maydis also requires invasion. Second, there is a correlationbetween genes that regulate agar invasion in Saccharomyces (S.)cerevisiae and genes that control invasion in pathogenic yeast. As S.cerevisiae is amenable to genetic studies, it can be utilized tomolecularly dissect the genetics of fungal invasion.

Homologs of certain S. cerevisiae genes required for invasion alsoregulate the production of secondary metabolites and secreted catabolicenzymes in other fungi. For example, activating mutations in Aspergillushomologs of the S. cerevisiae INV genes cause increased production ofthe secondary metabolite penicillin and a secreted alkaline phosphatase(Orejas et al., Genes Dev. 1995, 9:1622).

SUMMARY OF THE INVENTION

In one aspect, the invention features a method for determining whether acandidate compound decreases the expression of a gene operably linked toa fungal invasin gene promoter. The method generally includes the stepsof (a) providing a fungus expressing the gene operably linked to afungal invasin gene promoter; (b) contacting the fungus with thecandidate compound; and (c) detecting or measuring expression of thegene following contact of the fungus with the candidate compound. Inpreferred embodiments, the fungus is a wild-type strain (e.g.,Saccharomyces cerevisiae, Candida albicans, or Aspergillus nidulans); amutant strain; or a transgenic fungus. In other preferred embodiments,the gene used in the method of the invention is a fungal invasin gene.Exemplary fungal invasin genes include, without limitation, AFL1, DHH1,INV1, INV5, INV6, INV7, INV8, INV9, INV10, INV11, INV12, INV13, INV14,INV15, BEM2, CDC25, FLO11, IRA1, MCM1, MGA1, MUC1, PET9, PHD2, PHO23,PTC1, RIM15, SFL1, SRB11, SSD1, STE21, STP22, SW14, TPK2, TPK3, RIM1, orYPR1.

In still other preferred embodiments, the gene used in the method of theinvention is a reporter gene. Exemplary reporter genes useful in themethods of the invention include, without limitation, chloramphenicoltransacetylase (CAT), green fluorescent protein (GFP), β-galactosidase(lacZ), luciferase, URA3, or HIS3.

In preferred embodiments, the fungal invasin gene promoter utilized inthe methods of the invention is derived from the FLO11, MUC1, STA1, ST2,or STA3 gene promoter. In other preferred embodiments, the fungalinvasin gene promoter includes a promoter sequence derived from an AFL1,DHH1, INV1, INV5, INV6, INV7, INV8, INV9, INV10, INV11, INV12, INV13,RIM1, INV14, INV15, BEM2, CDC25, HOG1, IRA1, MCM1, MGA1, PET9, PHD2,PHO23, PTC1, RIM15, SFL1, SRB11, SSD1, STE21, STP22, SWI4, TPK2, TPK3,or YPR1 gene promoter. Preferably, the fungal invasin gene promoter is afragment or a deletion of the fungal invasin gene promoter (e.g., afragment of the FLO11 gene promoter); and, if desired, the fragment isfused to a basal promoter (e.g., a basal promoter from a PGK1, ADH1,GAL1-10, tet-R, MET25, CYC1 or CUP1 gene).

Typically, the expression of the gene (e.g., the endogenous FLO11 or arecombinant reporter gene expressed under the control of the FLO11 genepromoter or fragment thereof) is measured by assaying the RNA or proteinlevels or both of the expressed gene. For example, the polypeptideexpressed by the fungal invasin gene or by the reporter gene produces adetectable signal under conditions such that the compound causes ameasurable signal to be produced. Quantitatively determining the amountof signal produced requires comparing the amount of signal produced tothe amount of signal detected in the absence of any compound beingtested or upon contacting the cell with any other compound as isdescribed herein. The comparison permits the identification of thecompound as one which causes a change in the detectable signal producedby the expressed gene (e.g., at the RNA or protein level) and thusidentifies a compound that is capable of inhibiting fungal invasion. Adecrease in the expression of the fungal invasin gene is generallyaccompanied by an inhibition of fungal invasion or an inhibition of thedevelopmental switch from yeast form to pseudohyphal growth or both.

In related aspects, the invention also features a method for determiningwhether a candidate compound increases the expression of a gene operablylinked to a fungal invasin gene promoter. The method generally includesthe steps of (a) providing a fungus expressing the gene operably linkedto a fungal invasin gene promoter; (b) contacting the fungus with thecandidate compound; and (c) detecting or measuring expression of thegene following contact of the fungus with the candidate compound. Inpreferred embodiments, the method further includes determining whetherthe candidate compound increases the production of a secondarymetabolite in the fungus.

In another aspect, the invention features a method for determiningwhether a candidate compound inhibits fungal invasion. The methodgenerally includes the steps of (a) contacting a fungus with a candidatecompound under conditions suitable for invasion and (b) measuring ordetecting invasion by the fungus following contact with the candidatecompound. In preferred embodiments, the fungus is Candida albicans orSaccharomyces cerevisiae.

In another aspect, the invention features a method for determiningwhether a candidate compound promotes fungal invasion. The methodgenerally includes the steps of (a) contacting a fungus with a candidatecompound under conditions suitable for invasion and (b) measuring ordetecting invasion by the fungus following contact with the candidatecompound. In preferred embodiments, the fungus is Candida albicans,Saccharomyces cerevisiae, or Aspergillus nidulans.

In still another aspect, the invention features a method for identifyinga fungal invasion-promoting gene. The method generally includes thesteps of (a) expressing in a fungus (i) a first gene operably linked toa fungal invasin gene promoter and (ii) a second candidate gene or afragment thereof and (b) monitoring the expression of the first gene,wherein an increase in the expression of the first gene identifies thesecond candidate gene as a fungal invasion-promoting gene. In preferredembodiments, the fungus is a wild-type strain (e.g., Saccharomycescerevisiae, Aspergillus nidulans, Penicillium chrysogenum, or Acremoniumchrysogenum); is a mutant strain; or is a transgenic fungus.

Preferably, the first gene includes a fungal invasin gene (e.g., FLO11or MUC1). In yet other preferred embodiments, the first gene includes afungal invasin gene derived from AFL1, DHH1, INV1, INV5, INV6, INV7,INV8, INV9, INV10, INV11, INV12, INV13, INV14, INV15, BEM2, CDC25, HOG1,IRA1, RIM1, MCM1, MGA1, PET9, PHD2, PHO23, PTC1, RIM15, SFL1, SRB11,SSD1, STE21, STP22, SWI4, TPK2, TPK3, or YPR1 gene; or the first geneincludes a reporter gene (e.g., lacZ URA3, or HIS3).

In preferred embodiments, the fungal invasin gene promoter is derivedfrom the FLO11, MUC1, STA1, STA2, or STA3 gene promoter. In otherpreferred embodiments, the fungal invasin gene promoter is derived fromthe AFL1, DHH1, INV1, RIM1, INV5, INV6, INV7, INV8, INV9, INV10, INV11,INV12, INV13, INV14, INV 15, BEM2, CDC25, HOG1, IRA1, MCM1, MGA1, PET9,PHD2, PHO23, PTC1, RIM15, SFL1, SRB11, SSD1, STE21, STP22, SWI4, TPK2,TPK3, or YPR1 gene promoter; or is a fragment or a deletion of theabove-mentioned fungal invasin gene promoters. Preferably, the fragmentof a fungal invasin gene promoter is fused to a basal promoter (e.g., abasal promoter of a PGK1, ADH1, GAL1-10, tet-R, MET25, CYC1, or CUP1gene).

Preferably, the expression of the gene utilized in the method of theinvention is measured by assaying the protein level of the expressedfirst gene or by assaying the RNA level of the expressed first gene.

In related aspects, the invention also features a method for identifyinga fungal invasion-inhibiting gene. The method generally includes thesteps of (a) expressing in a fungus (i) a first gene operably linked toa fungal invasin gene promoter and (ii) a second candidate gene orfragment thereof and (b) monitoring the expression of the first gene,wherein a decrease in the expression of the first gene identifies thesecond candidate gene as a fungal invasion-inhibiting gene.

In yet another aspect, the invention features a method for increasingproduction of a secondary metabolite in a fungal cell; the methodgenerally includes the step of contacting the fungal cell with a fungalinvasion-promoting compound and culturing the cells under conditionswhich promote the increased synthesis of a secondary metabolite.

In another aspect, the invention features a method for increasingproduction of a secondary metabolite in a fungal cell. The methodgenerally includes the step of decreasing the expression of a fungalinvasion-inhibiting gene. In preferred embodiments, the decreasedexpression of the fungal invasion-inhibiting gene (e.g., HOG1, BEM2,RIM15, SFL1, IRA1, SSD1, SRB11, SWI4, or TPK3) results from aninactivation of the fungal invasion-inhibiting gene. In other preferredembodiments, the increased production of a secondary metabolite resultsfrom the expression of a mutated fungal invasion-inhibiting gene.

In still another aspect, the invention features a method for increasingproduction of a fungal secondary metabolite. The method generallyincludes the step of increasing the expression of a fungalinvasion-promoting gene. In preferred embodiments, the fungalinvasion-promoting gene is AFL1, DHH1, INV7, INV8, STE21, PET9, MEP2,INV1, INV5, INV6, INV9, INV10, INV11, INV12, INV13, INV14, INV15, CDC25,MCM1, MGA1, PHD2, PHO23, PTC1, RIM1, STP22, TPK2, or YPR1. In otherpreferred embodiments, the increased expression of the fungalinvasion-promoting gene is achieved by constitutively expressing thefungal invasion-promoting gene or by overexpressing such a gene. In yetother preferred embodiments, the fungal invasion-promoting gene ismutated.

In another aspect, the invention features a method for increasingproduction of a fungal secondary metabolite. The method generallyincludes the step of expressing a gene or fragment thereof that encodesan activated form of a invasion-promoting polypeptide. In preferredembodiments, the activated form of the invasion-promoting polypeptideincludes a fusion between the invasion-promoting polypeptide and asecond polypeptide that further enhances the activity of theinvasion-promoting polypeptide. In other preferred embodiments, the genehas a mutation.

In another aspect, the invention features a method for increasingproduction of a secondary metabolite in a fungal cell. The methodgenerally includes the step of decreasing the activity of a fungalinvasion-inhibiting polypeptide. In preferred embodiments, the fungalinvasion-inhibiting polypeptide has a mutation (e.g., adominant-inactive polypeptide). In other preferred embodiments, thefungal invasion-inhibiting polypeptide is Hog1, Bem2, Rim15, Ira1, Sfl1,Ssd1, Srb11, Swi4, or Tpk3.

In another aspect, the invention features a method for increasingproduction of a fungal secondary metabolite. The method generallyincludes the steps of increasing the activity of a fungalinvasion-promoting polypeptide. In preferred embodiments, the fungalinvasion-promoting polypeptide has a mutation (e.g., a dominant-activepolypeptide). In other preferred embodiments, the fungalinvasion-promoting polypeptide is Afl1, Dhh1, Inv1, Inv5, Inv6, Inv9,Inv10, Inv11, Inv12, Inv13, Inv14, Rim1,Inv15, Cdc25, Inv7, Mcm1, Mga1,Phd2, Pho23, Ptc1, Inv8, Ste2, Pet9, Mep2, Stp22, Ypr1.

In another aspect, the invention features a method of isolating a fungalinvasin gene. The method generally involves the steps of (a) providing afungus expressing a gene operably linked to a fungal invasin genepromoter; (b) mutagenizing the fungus; (c) measuring expression of thegene, wherein an increase or decrease in the expression of said geneidentifies a mutation in said invasin gene; and (d) using said mutationas a marker for isolating said invasin gene. In preferred embodiments,the fungus is a wild-type strain (e.g., Saccharomyces cerevisiae) or isa mutant strain. In preferred embodiments, the gene utilized in themethod includes a fungal invasin gene (e.g., FLO11, MUC1, AFL1, DHH1,INV1, INV5, INV6, INV7, INV8, INV9, INV10, INV11, INV12, INV13, INV14,INV15, BEM2, CDC25, HOG1, IRA1, MCM1, MGA1, PET9, PHD2, PHO23, RIM1,PTC1, RIM15, SFL1, SRB11, SSD1, STE21, STP22, SWI4, TPK2, TPK3, orYPR1). In other preferred embodiments, the gene includes a reporter gene(e.g., lacZ, URA3, or HIS3). In still other preferred, the fungalinvasin gene promoter is from FLO11, MUC1, STA1, STA2, or STA3 genepromoter; or is from the AFL1, DHH1, INV1, INV5, INV6, INV7, INV8, INV9,INV10, INV11, INV12, INV13, INV14, INV15, BEM2, CDC25, RIM1, HOG1, IRA1,MCM1, MGA1, PET9, PHD2, PHO23, PTC1, RIM15, SFL1, SRB11, SSD1, STE21,STP22, SWI4, TPK2, TPK3, or YPR1 gene promoter. In other preferredembodiments, the fungal invasin promoter is a fragment or deletion ofthe above-mentioned fungal invasin gene promoters. Preferably, thefragment of a fungal invasin gene promoter is fused to a basal promoter(e.g., a basal promoter of a PGK1, ADH1, GAL1-10, tet-R, MET25, CYC1 orCUP1 gene).

In another aspect, the invention features a method of using a cell(e.g., a fungal cell) for identifying a gene which regulates theexpression from a Candida albicans gene promoter. The method generallyincludes the steps of (a) providing a cell expressing a reporter geneoperably linked to a Candida albicans gene promoter; (b) expressing acandidate gene in the cell; and (c) detecting or measuring theexpression of the reporter gene. In preferred embodiments, the fungalcell is Saccharomyces cerevisiae.

In another aspect, the invention features a method for preparing atransgenic fungal cell having increased secondary metabolite production.The method generally includes the steps of (a) introducing a transgene(e.g., a transgene encoding an invasin gene such AFL1, DHH1, INV7, INV8,STE21, PET9, MEP2, INV1, INV5, INV6, INV9, INV10, INV11, RIM1, INV12,INV13, INV14, INV15, CDC25, MCM1, MGA1, PHD2, PHO23, PTC1, STP22, TPK2,YPR1 or a fragment thereof, which is positioned for expression in afungal cell) and (b) selecting a cell that expresses the transgene. Inpreferred embodiments, the transgene has a mutation (e.g., adominant-active mutation or a dominant-inactive mutation).

In another aspect, the invention features a method for increasing asecondary metabolite in a fungus. The method generally includes thesteps of (a) culturing the fungus in culture with conditions allowingfor secondary metabolite production; (b) adding to the culture a fungalinvasion-promoting compound; and (c) isolating the metabolite from theculture. In preferred embodiments, the fungus has a mutation. In otherpreferred embodiments, the fungus is a wild-type strain.

In still another aspect, the invention features a transgenic fungus(e.g., a filamentous fungus) which includes a mutation in an invasingene that inhibits its activity. Preferably, the invention features atransgenic filamentous fungus having a mutation in a HOG1, SWI4, BEM2,SRB11, SSD1, TPK3, SFL1, or an IRA1 gene or any combination thereof.Preferably, such mutations inhibit the activity of the expressed protein(e.g., Hog1, Swi4, Bem2, Srb 11, Ssd1, Tpk3, Sfl1, or Ira1 or anycombination thereof). In other preferred embodiments, the transgenicfungus has increase secondary metabolite production (e.g., increasedproduction of antibiotics).

In another aspect, the invention features a substantially pure Inv9polypeptide. Preferably, the Inv9 polypeptide is at least 55% identicalto the amino acid sequence of FIG. 6B (SEQ ID NO: 6). In preferredembodiments, the Inv9 polypeptide is from a fungus (e.g., a yeast suchas Saccharomyces). In other preferred embodiments, the Inv9 has invasionpromoting activities.

In a related aspect, the invention features an isolated nucleic acid(e.g., DNA) encoding an Inv9 polypeptide. Preferably, such an isolatednucleic acid sequence includes the INV9 gene of FIG. 6A (SEQ ID NO: 5)and complements an INV9 mutation in Saccharomyces cerevisiae.

In another aspect, the invention features a substantially pure Rim1polypeptide. Preferably, the Rim1 polypeptide is at least 75% identicalto the amino acid sequence of FIG. 5A (SEQ ID NO: 4) in the zinc-fingerdomain, and at least 25% identical throughout the entire length of thepolypeptide amino acid sequence. In preferred embodiments, the Rim1polypeptide is from a fungus (e.g., a yeast such as Saccharomyces). Inother preferred embodiments, the Rim1 polypeptide has invasion promotingactivities.

In a related aspect, the invention features an isolated nucleic acid(e.g., DNA) encoding a Rim1 polypeptide. Preferably, such an isolatednucleic includes the RIM1 gene of FIG. 5A (SEQ ID NO: 3) and complementsa RIM1 mutation in Saccharomyces cerevisiae, as is described herein.

In related aspects, the invention further features a cell or a vector(for example, a fungal expression vector), each of which includes anisolated nucleic acid molecule of the invention. In preferredembodiments, the cell is a fungal cell (for example, S. cerevisiae). Inyet another preferred embodiment, the isolated nucleic acid molecule ofthe invention is operably linked to a promoter that mediates expressionof a polypeptide encoded by the nucleic acid molecule. The inventionfurther features a cell (for example, a fungal cell) which contains thevector of the invention.

In still another aspect, the invention features a transgenic fungusincluding any of the above nucleic acid molecules of the invention,wherein the nucleic acid molecule is expressed in the transgenic fungus.

In related aspects, the invention also features a method of producing anInv9 or Rim1 polypeptide. The method involves: (a) providing a celltransformed with a nucleic acid molecule of the invention positioned forexpression in the cell; (b) culturing the transformed cell underconditions for expressing the nucleic acid molecule; and (c) recoveringthe Inv9 or Rim1 polypeptide. The invention further features arecombinant Inv9 or Rim1 polypeptide produced by such expression of anisolated nucleic acid molecule of the invention, and a substantiallypure antibody that specifically recognizes and binds to each of thesepolypeptides or a portion thereof.

By “fungal invasion” is meant a process by which a fungus penetrates,digs, adheres to, or attaches to a substrate. Invasion of a substrate bya fungus may be measured according to standard methods as describedherein.

By “fungal invasin” gene is meant a gene encoding a polypeptide capableof promoting or inhibiting the invasion by a fungus into a substrate.This response may occur at the transcriptional level or it may beenzymatic or structural in nature. Fungal invasin genes may beidentified and isolated from any fungal species, using any of thesequences disclosed herein in combination with conventional methodsknown in the art.

By “polypeptide” is meant any chain of amino acids, regardless of lengthor post-translational modification (for example, glycosylation orphosphorylation).

By a “reporter gene” is meant a gene whose expression may be assayed;such genes include, without limitation, genes encoding β-galactosidase,β-glucosidase, β-glucosidase, and invertase, amino acid biosyntheticgenes, e.g., the yeast LEU2, HIS3, LYS2, TRP1 genes, nucleic acidbiosynthetic genes, e.g., the yeast URA3 and ADE2 genes, the mammalianchloramphenicol transacetylase (CAT) gene, or any surface antigen genefor which specific antibodies are available. A reporter gene may encodea protein detectable by luminescence or fluorescence, such as greenfluorescent protein (GFP). Reporter genes may encode also any proteinthat provides a phenotypic marker, for example, a protein that isnecessary for cell growth or viability, or a toxic protein leading tocell death, or the reporter gene may encode a protein detectable by acolor assay leading to the presence or absence of color. Alternatively,a reporter gene may encode a suppressor tRNA, the expression of whichproduces a phenotype that can be assayed. A reporter gene according tothe invention includes elements (e.g., all promoter elements) necessaryfor reporter gene function.

By “substantially identical” is meant a polypeptide or nucleic acidexhibiting at least 25%, preferably 50%, more preferably 80%, and mostpreferably 90%, or even 95% identity to a reference amino acid sequence(for example, the amino acid sequence shown in FIG. 5A (SEQ ID NO: 4) ornucleic acid sequence (for example, the nucleic acid sequences shown inFIG. 5A; SEQ ID NO: 3). For polypeptides, the length of comparisonsequences will generally be at least 16 amino acids, preferably at least20 amino acids, more preferably at least 25 amino acids, and mostpreferably 35 amino acids or greater. For nucleic acids, the length ofcomparison sequences will generally be at least 50 nucleotides,preferably at least 60 nucleotides, more preferably at least 75nucleotides, and most preferably 110 nucleotides or greater.

Sequence identity is typically measured using sequence analysis software(for example, Sequence Analysis Software Package of the GeneticsComputer Group, University of Wisconsin Biotechnology Center, 1710University Avenue, Madison, Wis. 53705, BLAST, BEAUTY, orPILEUP/PRETTYBOX programs). Such software matches identical or similarsequences by assigning degrees of homology to various substitutions,deletions, and/or other modifications. Conservative substitutionstypically include substitutions within the following groups: glycinealanine; valine, isoleucine, leucine; aspartic acid, glutamic acid,asparagine, glutamine; serine, threonine; lysine, arginine; andphenylalanine, tyrosine.

By a “substantially pure polypeptide” is meant a polypeptide (forexample, an invasin polypeptide such as the Inv9 or Rim1 polypeptide)that has been separated from components which naturally accompany it.Typically, the polypeptide is substantially pure when it is at least60%, by weight, free from the proteins and naturally-occurring organicmolecules with which it is naturally associated. Preferably, thepreparation is at least 75%, more preferably at least 90%, and mostpreferably at least 99%, by weight, an invasin polypeptide. Asubstantially pure invasin polypeptide may be obtained, for example, byextraction from a natural source (for example, a fungal cell); byexpression of a recombinant nucleic acid encoding an invasinpolypeptide; or by chemically synthesizing the protein. Purity can bemeasured by any appropriate method, for example, column chromatography,polyacrylamide gel electrophoresis, or by HPLC analysis.

By “derived from” is meant isolated from or having the sequence of anaturally-occurring sequence (e.g., a cDNA, genomic DNA, synthetic, orcombination thereof).

By “isolated DNA” is meant DNA that is free of the genes which, in thenaturally-occurring genome of the organism from which the DNA of theinvention is derived, flank the gene. The term therefore includes, forexample, a recombinant DNA that is incorporated into a vector; into anautonomously replicating plasmid or virus; or into the genomic DNA of aprokaryote or eukaryote; or that exists as a separate molecule (forexample, a cDNA or a genomic or cDNA fragment produced by PCR orrestriction endonuclease digestion) independent of other sequences. Italso includes a recombinant DNA which is part of a hybrid gene encodingadditional polypeptide sequence.

By “transgenic fungal cell” is meant a fungal cell into which (or intoan ancestor of which) has been introduced, by means of recombinant DNAtechniques, a DNA molecule encoding (as used herein) an invasinpolypeptide.

By “positioned for expression” is meant that the DNA molecule ispositioned adjacent to a DNA sequence which directs transcription andtranslation of the sequence (i.e., facilitates the production of, forexample, an invasin polypeptide, a recombinant protein, or an RNAmolecule).

By “promoter sequence” is meant any minimal sequence sufficient todirect transcription. Included in the invention are promoter elementsthat are sufficient to render promoter-dependent gene expressioncontrollable for gene expression, or elements that are inducible byexternal signals or agents; such elements may be located in the 5′ or 3′regions of the native gene or engineered into a transgene construct.

By “operably linked” is meant that a gene and a regulatory sequence(s)are connected in such a way as to permit gene expression when theappropriate molecules (for example, transcriptional activator proteins)are bound to the regulatory sequence(s).

By “candidate gene” is meant any piece of DNA which is inserted byartifice into a cell and expressed in that cell. A candidate gene mayinclude a gene which is partly or entirely heterologous (i.e., foreign)to the transgenic organism, or may represent a gene homologous to anendogenous gene of the organism.

By “transgene” is meant any piece of DNA which is inserted by artificeinto a cell, and becomes part of the genome of the organism whichdevelops from that cell. Such a transgene may include a gene which ispartly or entirely heterologous (i.e., foreign) to the transgenicorganism, or may represent a gene homologous to an endogenous gene ofthe organism.

By “transgenic” is meant any cell which includes a DNA sequence which isinserted by artifice into a cell and becomes part of the genome of theorganism which develops from that cell. As used herein, the transgenicorganisms are generally transgenic fungal cells. A transgenic fungalcell according to the invention may contain one or more invasin genes.

By “increasing production of a secondary metabolite” is meant a greaterlevel of production of a secondary metabolite in a transgenic fungus ofthe invention than the level of production relative to a control fungus(for example, a non-transgenic fungus). In preferred embodiments, thelevel of secondary metabolite production in a transgenic fungus of theinvention is at least 10% greater (and preferably more than 30% or 50%)than the resistance of a control fungus. The level of secondarymetabolite production is measured using conventional methods.

By “detectably-labelled” is meant any direct or indirect means formarking and identifying the presence of a molecule, for example, anoligonucleotide probe or primer, a gene or fragment thereof, or a cDNAmolecule or a fragment thereof. Methods for detectably-labelling amolecule are well known in the art and include, without limitation,radioactive labelling (for example, with an isotope such as ³²P or ³⁵S)and nonradioactive labelling (for example, chemiluminescent labelling,for example, fluorescein labelling).

By “purified antibody” is meant antibody which is at least 60%, byweight, free from proteins and naturally-occurring organic moleculeswith which it is naturally associated. Preferably, the preparation is atleast 75%, more preferably 90%, and most preferably at least 99%, byweight, antibody, for example, an acquired resistancepolypeptide-specific antibody. A purified invasin antibody (e.g. Inv9 orRim1) may be obtained, for example, by affinity chromatography using arecombinantly-produced acquired resistance polypeptide and standardtechniques.

By “specifically binds” is meant an antibody which recognizes and bindsan invasin polypeptide but which does not substantially recognize andbind other molecules in a sample, for example, a biological sample,which naturally includes an invasin polypeptide such as Inv9 or Rim1.

By a “mutation” is meant an alteration in sequence, either bysite-directed or random mutagenesis. A mutated form of a proteinencompasses point mutations as well as insertions, deletions, orrearrangements. A mutant is an organism containing a mutation

The invention provides long awaited advantages over a wide variety ofstandard screening methods used for distinguishing and evaluating theefficacy of a compound in regulation of gene expression in fungalpathogens. For example, the methods allow for the identification, bygenetic selection in a high throughput format, of peptides and compoundsthat specifically activate or inhibit fungal invasion. These methodsalso allow the mode of action for such agents to be rapidly delineated.Moreover, these methods are amenable to an iterative compoundmodification and retesting process to allow for the evolution of moreeffective compounds from initial hits and leads.

Compounds which inhibit fungal invasion will likely also prevent fungalvirulence. These compounds may have therapeutic value in treating plantor animal fungal diseases. Compounds which promote fungal invasion maybe useful in increasing yields of commercially important fungalsecondary metabolites.

The invention also provides an approach to isolating novel fungal genesimportant for pathogenesis. These novel genes and the proteins theyencode comprise additional targets for compounds.

In addition, the invention provides a means to increase the yield ofcommercially important secondary metabolites by genetic manipulation ofthe fungal organism itself. This facilitates the large scale productionof fungal products which to date has not been possible. In addition, itallows for the facile identification of “potentiators,” (i.e., compoundsand peptides) that can activate secondary metabolite production whencontacting, or expressed in, fungi. The ability to increase fungalsecondary metabolite production has at least two important applications.First, increasing production of secondary metabolites will facilitateidentification of new antimicrobial compounds in fungi that otherwisemake undetectable levels of these compounds in the laboratory. Second,it will allow increased production of existing secondary metaboliteswhich are useful clinically or for research.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

DETAILED DESCRIPTION

The drawings will first be described.

Drawings

FIG. 1 is a western blot showing the effect of mutations in the INVpathway and STE pathway on Inv8 proteolytic processing.

FIG. 2A is a series of photographs showing haploid invasion phenotypesfor wild-type strains and strains with mutations in TPK genes.

FIG. 2B is a series of photographs showing pseudohyphal phenotypes forwild-type strains and strains with mutations in TPK genes.

FIG. 3 is a photograph showing the results of a yeast two-hybridanalysis showing preferential affinity of Tpk2 for Mga1 and Sfl1. Geneproducts tested are shown on the vertical and horizontal axes. Yeastgrowth indicates an interaction between the products of the genes shownon the respective axes.

FIG. 4A is a photograph of an epistasis analysis slowing that FLO11 actsdownstream of both SFL1 and TPK2 during haploid invasive growth.

FIG. 4B is a photograph of an epistasis analysis showing that FLO11 actsdownstream of both SFL1 and TPK2 during pseudohyphal growth.

FIGS. 5A-1 to 5A-12 show the DNA sequence of the Candida RIM1 gene (SEQID NO: 3) and the predicted encoded protein (SEQ ID NO: 4).

FIG. 5B is a schematic illustration describing the complementation ofthe S. cerevisiae inv8 phenotype with Candida RIM1.

FIGS. 6A-1 to 6A-3 show the DNA sequence of S. cerevisiae INV9 (SEQ IDNO: 5).

FIG. 6B shows the predicted amino acid sequence of S. cerevisiae Inv9(SEQ ID NO: 6).

FIG. 7 is a series of photographs of phenotypic analysis of the serumresponse of Candida rim1/rim1 mutants.

FIG. 8 is a series of photographs showing that processing of Inv8 isrequired for its nuclear accumulation.

FIG. 9 is a photograph of a northern blot showing the steady-statelevels of FLO11 mRNA in a variety of invasion-defective (flo8, ste12,phd1, phd2, afl1, ras2, tpk2, tec1, flo11, and inv6) and hyper-invasivemutants (ira1). The blot was probed with probes for FLO11 and ACT1.

FIG. 10 is a schematic illustration of genes and pathways that regulateinvasion. Pointed arrows denote activation, flat-ended arrows indicaterepression.

FIG. 11 is a schematic illustration showing the relative positions ofthe FLO11 promoter fragments used to generate reporter constructs.

FIG. 12 shows the results of a gradient plate assay, demonstrating thatan agar invasion screen could be employed as a system for identifyingantifungal compounds.

We have discovered a series of interconnected signal transductioncascades which regulate fungal invasion and pseudohyphal growth. From S.cerevisiae, we identified new roles for previously characterized genesinvasion and pseudohyphal growth (AFL1, DHH1, INV1, INV5, INV6, INV7,INV8, INV9, INV10, INV11, INV12, INV13, INV14, INV15, BEM2, CDC25, HOG1,IRA1, MCM1, MGA1, PET9, PHD2, PHO23, PTC1, RIM15, SFL1, SRB11, SSD1,STE21, STP22, SWI4, TPK2, TPK3, YPR1). Genetic and biochemical analysishas allowed us to place these genes, and other genes known to regulateinvasion, into a series of parallel signal transduction cascades. Table1, below, lists the genes of which we have identified that regulateinvasion and/or pseudohyphal growth in S. cerevisiae. These genes arecross-referenced with the corresponding sequence name as designated bythe Saccharomyces Genome Database. Sequences of these genes and theirputative transcriptional regulatory elements are also found at theSaccharomyces Genome Database(http://genome-www.stanford.edu/Saccharomyces/).

TABLE 1 Sequence Gene Name Sequence Name Gene Name Name INVI/VPS36YLR417w PHD1 YKL043w INV3/DFG16 YOR030w ELM1 YKL048c INV5 YOR275c FLO11YIR019c INV6 YMR164c SSD1 YDR293c INV7 YDL233w IRA1 YBR140c INV8 YHL027wCDC25 YLR310c INV9 YGL046w + YGL045w RAS2 YNL098c INV10 YMR063w BCY1YIL033c INV11 YNL294c TPK3 YKL166c INV12/SNF8 YPL002c MGA1 YGR249w INV13YJR102c SFL1 YOR140w INV14 YMR154c BEM2 YER155c INV15/VPS28 YPL065wCDC55 YGL190c INV16 not cloned NPR1 YNL183c STP22/ YCL008c MEP2 YNL142wMCM1 YMR043w SHR3 YDL212w FLO8 YER108c URE2 YNL229c WH13 YNL197c GLN3YER040w SRV2 YNL138w CDC42 YLR229c CYR1 YJL005w CDC24 YAL041w AFL1YEL007w STSE11 YLR362w PTC YDL006w STE7 YDL159w STE20 YHL007c KSS1YGR040w TPK2 YPL203w STE12 YHR084w YPR1 YDR368w TEC1 YBR083w PH023YNL097c STE21 YDR335w DHH1 YDL160c SIR4 YDR227w PHD2 YOL116w PET9YBL030c GRR1 YJR090c SWI4 YER111c RIM15 YFL033c SRB11 YNL025c HOG1YLR113w

We have constructed a “wiring diagram” of these signal transductioncascades and shown directly that many of these genes regulate fungalinvasion, at least in part, through the regulation of a single gene,FLO11. The identification of the convergence of signals onto a singletarget has facilitated improved screening methods designed to evaluateand identify therapeutic agents useful for inhibiting fungalpathogenesis in either animal or plant hosts. Furthermore, our discoveryprovides the basis for screening methods useful for identifying avariety of new fungal virulence factors. Identification of suchvirulence factors further facilitates the development of targetedreagents for use as anti-pathogens. In addition, the improved screeningmethods provide the basis for identifying factor that increaseproduction of important fungal secondary metabolites.

The following experimental examples are intended to illustrate, notlimit, the scope of the claimed invention.

Screens to Isolate Genes Involved in Pseudohyphal Growth and Invasion

Below we describe experimental evidence that a series of signaltransduction cascades operates in cooperation to regulate pseudohyphalgrowth and haploid digging in solid medium in S. cerevisiae.

On solid media containing high glucose and low nitrogen, diploid cellsof S. cerevisiae form pseudohyphae, which consist of chains of elongatedcells that form invasive filaments (Gimeno et al., Cell 1992,68:1077-1090). On rich medium, haploid cells, but not diploid cells,manifest invasive growth, referred to as “digging” (Roberts and Fink,Genes Dev. 1994, 8:2974-85). The nutritional signals that eventuate inboth pseudohyphal growth and haploid invasive growth involve severalpathways. To isolate S. cerevisiae genes involved in these behaviors, wehave performed a series of screens for mutants defective in eitherpseudohyphal growth or haploid invasion. These screens are describedbelow.

Screen 1. Identification of Mutants that Eliminate Invasion DuringPseudohyphal Growth.

Mutagenesis of yeast and screens were conducted as described in Möschand Fink, (Genetics 1997, 145:671-684). Briefly, a direct visual screenfor transposon-induced mutants unable to invade the agar when grown onlow nitrogen SLAD agar was conducted in a MATa/α haploid strain capableof pseudohyphal growth. Secondary screening identified a subset ofmutants that make long cells and, thus, are capable of all aspects ofpseudohyphal growth other than invasion.

Screen 2. Identification of Digging-Defective Mutants.

S. cerevisiae mutants defective for haploid invasive growth wereidentified using a plate washing assay described in (Roberts and Fink,supra). Transposon-mutagenized haploids, constructed as described inMösch and Fink (Genetics 1997, 145:671-84 and Burns et al., Genes Dev.1994 8:1087-105), were plated on YPD to allow formation of well-isolatedsingle colonies. After 3-5 days growth at 30° C., plates werereplica-plated and the surface of the master plate was washed with astream of water. Colonies of invasion-defective mutants wash completely(or nearly so) from the agar, whereas many cells of a wild-type strainremained in the agar after washing. Mutant strains were subsequentlypicked from the replicas of colonies that exhibited aninvasion-defective phenotype.

Screen 3. Identification of Hyperinvasive Mutants.

Transposon-mutagenized haploids were plated to YPD and grown asdescribed in Screen 2 above. Colonies with a surface morphology thatappeared more “rough” than wild-type colonies were picked—a rough colonysurface is indicative of hyperinvasion. Secondary testing subsequentlyidentified mutants in which more cells remained in the agar afterwashing as compared to wild-type controls. Such mutants were retained ashyper-invasive mutants.

Screen 4. Identification of Mutations Specifically Affecting The INVPathway.

This screen relied upon our observation that, unlike mutations affectingother characterized genes required for pseudohyphal growth, inv mutantswere more resistant than wild-type strains to the whitening agentcalcoflour. In addition, as is true for all invasion-defective mutants,inv haploids exhibited a colony surface morphology that appears moresmooth than wild-type colonies when grown on YPD agar. A collection oftransposon-induced calcoflour-resistant mutants exhibiting a smoothcolony morphology when grown on YPD agar were also identified.

For all mutations induced by transposon insertion, genomic DNA flankingthe transposon insertion site was isolated and sequenced by conventionalmethods. Nucleotide sequence of flanking DNA was then compared to thegenomic sequence of S. cerevisiae to identify the affected gene.

Screen 5. Identification of High-Copy Suppressors of anInvasion-Defective Mutant.

A library of plasmids containing yeast genomic DNA was transformed intoan invasion-defective (Inv-) diploid whi3 strain. These library plasmidscontained an origin of replication that promotes maintenance inhigh-copy number (the 2 μ origin). Transformants were plated onselective media (SC-URA), plates were incubated for 1-2 days, and then aplate washing assay was performed. High-copy plasmids that suppress theInv-phenotype promoted a transient period of invasive growth intosynthetic media. This growth was identified as small pits of coloniesthat remained in the agar after the plate washing assay. Several geneswere identified as high-copy suppressors of the Inv-phenotype of whi3mutants, including MCM1 and PHD2. Subsequent analysis of phd2-deletedand mcm1 partial loss-of-function mutants demonstrated that these genesregulate invasion and FLO11 expression.

From the above screens, we have identified 60 genes which regulatepseudohyphal growth or haploid invasion in S. cerevisiae. These genes,referred to as invasins, have been further categorized into at least sixsignal transduction cascades. Based on the resulting phenotype followingmutation or overexpression, these genes can be further classified as“fungal invasion-promoting” or “fungal invasion-inhibiting.” Forexample, a S. cerevisiae strain containing a complete deletion of AFL1does not form filaments or invade under conditions in which a wild-typestrain does, and no invasion is observed under conditions that normallyinduced invasion during pseudohyphal growth or haploid digging. Anexample of a signal transduction cascade is described below

Twelve S. cerevisiae genes which comprise this signal transductionpathway required for agar invasion have been identified, cloned, andcharacterized. INV8, encoding a transcription factor whose activity isregulated by proteolysis, is the most downstream member of the pathway.The other twelve members (INV1, INV3, INV5, INV9, INV10, INV11, INV12,INV13, INV14, INV15, INV16, and STP22) are all required for proteolyticregulation of Inv8. As shown in FIG. 1, mutations in these genes, butnot mutations in any other invasin genes thus far examined, block Inv8processing. One of the INV members, Inv14, is a cytoplasmic cysteineprotease of the calpain family. It was surprising that this signaltransduction pathway was conserved in fungi and served, in other fungi,to regulate the production of secondary metabolites and secreted enzymesthat are able to degrade extracellular substrates. Homologs of the S.cerevisiae genes have been found, for example, in A. nidulans, where asimilar set of genes regulate transcription of a number of secretedcatabolizing enzymes in response to extracellular pH; in Yarrowialipolytica, as the transcriptional regulator of an abundant, secretedalkaline protease; and in Penicillium (P.) chrysogenum and A. niger,where it has a role similar to that found in A. nidulans. In A. nidulansand in P. chrysogenum, the pathway directly regulates the production ofpenicillin (penicillin production is regulated by the activated form ofthe transcription factor).

Other additional members of these signal transduction cascades areidentified through a variety of standard techniques. First, enhancersand suppressors of phenotypes of mutant strains are isolated usingstandard techniques known to those skilled in the art (Ausubel, supra).Second, proteins which interact with pathway members are isolated usingyeast two-hybrid methods (Fields and Song, Nature 1989, 340:245-246;Chien et al., Proc. Natl. Acad. Sci. USA 1991, 88:9578-9582; Brent andFinley, Annu. Rev. Genet. 1997, 31:663-704). In one example, proteinswhich specifically interact with an A kinase catalytic subunit Tpk2, andnot with related proteins Tpk1 and Tpk3 were identified and shown to beimportant for pseudohyphal development. Diploid tpk2 strains grown onSLAD (low nitrogen medium) were completely defective for pseudohyphaldevelopment, whereas tpk3 diploid strains were greatly enhanced forfilamentation (FIG. 2B). Similarly, haploid tpk2 mutants were defectivefor invasive growth and tpk3 mutants were hyperinvasive (FIG. 2A). tpk1mutants were indistinguishable from wild-type strains as both haploidsand diploids. The accentuated filamentation and invasion phenotypes oftpk3 mutants required a functional Tpk2 as tpk2 tpk3 mutants had thesame phenotype as tpk2 single mutants. As Tpk2 appeared to be uniquelyrequired for pseudohyphal growth, we used a two-hybrid screen toidentify proteins that interact specifically with Tpk2. The intact openreading frame of TPK2 was fused to coding sequence for the Ga14 DNAbinding domain and used to screen a library of yeast genomic fragmentsfused to coding sequence for the Ga14 transcriptional activation domain.Activation of the GAL4-ADE2 reporter, leading to adenine-independentgrowth of an ade-strain, was taken as preliminary evidence for aninteraction between the Ga14 DNA binding domain:Tpk2 fusion and the Tpk2interacting protein fused to the Ga14 activation domain. Positive cloneswere then retested in the two-hybrid assay with Tpk1, Tpk2, Tpk3, orDph1 fused to the Ga14 DNA binding domain. DPH1, a gene required fordiphthamide biosynthesis, was used to control for interactions that werenot specific to the PKA isoforms.

Two classes of proteins interacted with Tpk2 in the two-hybrid assay:the first class was indiscriminate and interacted with Tpk1, Tpk2, andTpk3; the second class was selective and interacted preferentially withTpk2 (FIG. 3). One of the proteins that interacted with all threecatalytic subunits is Bcy1, the negative regulatory subunit of PKA,which had previously been shown to bind Tpk1, Tpk2, and Tpk3. The secondclass consisted of Sfl1 and Mga1, putative transcription factorsbelonging to a group of five yeast proteins (Mga1, Sfl1, Hsf1, Skn7, andHms2) containing a helix-turn-helix DNA binding motif. All but Skn7contain at least one consensus PKA phosphorylation site([R/K][R/K]X[S/T]): Sfl1 has five, Mga1 has two, Hsf1 has four, and Hms2has one.

We deleted the open reading frame of both MGA1 and SFL1. sfl1 mutantshad a dramatic phenotype: sfl1 haploid strains were extremely flocculentand hyperinvasive, whereas diploids homozygous for a deletion of sfl1were greatly enhanced for filamentation (FIG. 4A-B). We constructed ansfl1 tpk2 double mutant to see if the tpk2 pseudohyphal defect wasblocked by loss of function of Sfl1. sfl1 tpk2 double mutants had thesame phenotype as sfl1 single mutants, suggesting Sfl1 acts downstreamof Tpk2 (FIG. 4A-B). We could not detect a dramatic defect inpseudohyphal formation in mga1 null mutants.

Similar two-hybrid screens can also be used to identify additionalmembers of any of the signal transduction cascades described herein.Candidate genes are then analyzed using a variety of techniques known tothose skilled in the art, such as construction of mutant strains oroverexpression.

Identification of transcriptional targets of genes in the fungal signaltransduction pathways, such as Inv8 or Sfl1, are used to identify geneproducts which are directly responsible for extracellular substratedegradation or invasion. For example, identification of Inv8transcriptional targets is useful to identify gene products directlyresponsible for agar invasion. Identification of such target genesprovides excellent reporters of the INV pathway, which will make itpossible to demonstrate the relative activities of the unprocessedversus the proteolyzed Inv8 protein and to define the mechanisms bywhich Inv8 activates and represses target genes. Alternatively,identification of homologs, such as by screening for complementation ofthe phenotype in S. cerevisiae, in other fungi can be carried out toidentify fungal gene products which regulate production of secondarymetabolites, such as antibiotics, anti-hypercholesterolemic agents,immunosuppressives, or antifungal drugs.

Based on the work described herein, it is possible to discover anddevelop novel antifungal therapeutic drugs; identify and commercializenew fungal secondary metabolites; improve yields of presently availablefungal products; and develop technologies and products to address unmetfungal challenges. It is likely that the signal transduction machineryis conserved among fungi. Thus, based on the discoveries describedherein, each of these signal transduction cascades represents a targetfor antifungal drugs and/or regulation of secondary metabolites. Strainsof S. cerevisiae carrying mutant alleles of any of the genes can be usedto screen for fungal homologs, including those from important pathogenicfungi and commercially important fungi, such as Aspergillus sp.,Penicillium sp., Acremonium chrysogenum, Yarrowia lipolytica and Phaffiarhodozyma, which are capable of complementing or rescuing the mutantphenotype. These strains can be genetically modified such that therescued organisms are capable of increased growth or survival, such thatthese organisms can be isolated using selection based screens describedherein. Selection-based screens allow for high-throughput, and thusprovide a more rapid approach to gene isolation than those currentlyused. Moreover, screens for genes which complement mutant phenotypesallows for isolation of genes which share functional properties butwhich do not contain high degrees of similarity at the nucleotide oramino acid level.

Isolation of Invasin Homologs

Any fungal cell can serve as the nucleic acid source for the molecularcloning of an invasin homolog. Isolation of an invasin homolog (e.g.,AFL1, DHH1, INV1, INV5, INV6, INV7, INV8, INV9, INV10, INV11, INV12,INV13, INV14, INV15, BEM2, CDC25, HOG1, IRA1, MCM1, MGA1, PET9, PHD2,PHO23, PTC1, RIM1, RIM15, SFL1, SRB11, SSD1, STE21, STP22, SWI4, TPK2,TPK3, or YPR1) involves the isolation of those DNA sequences whichencode a polypeptide exhibiting properties or activities associated withpromotion or inhibition of fungal invasion. Based on the sequences ofthe invasin genes and polypeptides described herein, the isolation ofadditional invasin homolog regulatory and coding sequences from avariety of fungi (e.g.,Candida, Aspergillus, Penicillium, Mucor,Monascus, Trichoderma, Fusarium, Tolypocladum, Acremonium, Cryptococcus,Ustilago, Magneporthe, Acremonium, Yarrowia, and Phaffia) is madepossible using standard strategies and techniques that are well known inthe art.

In one particular example, any of the invasin sequences described hereinmay be used, together with conventional screening methods of nucleicacid hybridization screening. Such hybridization techniques andscreening procedures are well known to those skilled in the art and aredescribed, for example, in Benton and Davis, Science 1977, 196:180;Grunstein and Hogness, Proc. Natl. Acad. Sci., USA 1975, 72:3961;Ausubel et al. et al., 1997, Current Protocols in Molecular Biology,Wiley Interscience, New York; Berger and Kimmel, Guide to MolecularCloning Techniques, 1987, Academic Press, New York; and Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, New York. In one particular example, all or part of the Candidaalbicans RIM1 cDNA (described herein) may be used as a probe to screen arecombinant fungus DNA library (e.g., a recombinant expression libraryprepared from Aspergillus nidulans) for homologs having sequenceidentity to the Candida albicans RIM1 gene. Hybridizing sequences aredetected by plaque or colony hybridization according to the methodsdescribed below.

Alternatively, using all or a portion of the amino acid sequence of theRim1 polypeptide, one may readily design Rim1-specific oligonucleotideprobes, including Rim1 degenerate oligonucleotide probes (i.e., amixture of all possible coding sequences for a given amino acidsequence). These oligonucleotides may be based upon the sequence ofeither DNA strand and any appropriate portion of the Rim1 sequence.General methods for designing and preparing such probes are provided,for example, in Ausubel et al. (supra), and Berger and Kimmel (supra).These oligonucleotides are useful for RIM1 homolog isolation, eitherthrough their use as probes capable of hybridizing to RIM1 complementarysequences or as primers for various amplification techniques, forexample, polymerase chain reaction (PCR) cloning strategies. If desired,a combination of different oligonucleotide probes may be used for thescreening of a recombinant DNA library. The oligonucleotides may bedetectably-labeled using methods known in the art and used to probefilter replicas from a recombinant DNA library. Recombinant DNAlibraries are prepared according to methods well known in the art, forexample, as described in Ausubel et al. (supra), or they may be obtainedfrom commercial sources.

In one particular example of this approach, homolog RIM1 sequenceshaving greater than 75% identity are detected or isolated using highstringency conditions. High stringency conditions may includehybridization at about 42° C. and about 50% formamide, 0.1 mg/mL shearedsalmon sperm DNA, 1% SDS, 2×SSC, 10% Dextran sulfate, a first wash atabout 65° C., about 2×SSC, and 1% SDS, followed by a second wash atabout 65° C. and about 0.1×SSC. Alternatively, high stringencyconditions may include hybridization at about 42° C. and about 50%formamide, 0.1 mg,/mL sheared salmon sperm DNA, 0.5% SDS, 5×SSPE,1×Denhardt's, followed by two washes at room temperature and 2×SSC, 0.1%SDS, and two washes at between 55-60° C. and 0.2×SSC, 0.1% SDS.

In another approach, low stringency hybridization conditions fordetecting RIM1 homologs having about 25% or greater sequence identity tothe Candida albicans RIM1 gene described herein include, for example,hybridization at about 42° C. and 0.1 mg/mL sheared salmon sperm DNA, 1%SDS, 2×SSC, and 10% Dextran sulfate (in the absence of formamide), andawash at about 37° C. and 6×SSC, about 1% SDS. Alternatively, the lowstringency hybridization may be carried out at about 42° C. and 40%formamide, 0.1 mg/mL sheared salmon sperm DNA, 0.5% SDS, 5×SSPE, 1×Denhardt's, followed by two washes at room temperature and 2×SSC, 0.1%SDS and two washes at room temperature and 0.5×SSC, 0.1% SDS. Thesestringency conditions are exemplary; other appropriate conditions may bedetermined by those skilled in the art.

If desired, RNA gel blot analysis of total or poly(A+) RNAs isolatedfrom any fungus (e.g., those fungi described herein) may be used todetermine the presence or absence of a RIM1 transcript usingconventional methods. As an example, a northern blot of Aspergillusnidulans RNA is prepared according to standard methods and probed with aRIM1 gene fragment in a hybridization solution containing 50% formamide,5×SSC, 2.5×Denhardt's solution, and 300 μg/mL salmon sperm DNA at 37° C.Following overnight hybridization, the blot was washed two times for tenminutes each in a solution containing 1×SSC, 0.2% SDS at 37° C. Anautoradiogram of the blot is used to demonstrate the presence anRIM1-hybridizing RNA in the fungal RNA sample. A hybridizing band istaken as an indication that this fungus expresses a C. albicans RIM1homolog. Isolation of this hybridizing transcript is performed usingstandard cDNA cloning techniques. Other fungal invasions may beevaluated and cloned in a similar fashion.

As discussed above, invasin oligonucleotides (e.g., oligonucleotidesprepared from the Candida albicans RIM1 gene) may also be used asprimers in amplification cloning strategies, for example, using PCR. PCRmethods are well known in the art and are described, for example, in PCRTechnology, Erlich, ed., Stockton Press, London, 1989; PCR Protocols: AGuide to Methods and Applications, Innis et al., eds., Academic Press,Inc., New York, 1990; and Ausubel et al. (supra). Primers are optionallydesigned to allow cloning of the amplified product into a suitablevector, for example, by including appropriate restriction sites at the5′ and 3′ ends of the amplified fragment (as described herein). Ifdesired, RIM1 sequences may be isolated using the PCR “RACE” technique,or Rapid Amplification of cDNA Ends (see, e.g., Innis et al. (supra)).By this method, oligonucleotide primers based on an Candida albicansRIM1 sequence are oriented in the 3′ and 5′ directions and are used togenerate overlapping PCR fragments. These overlapping 3′- and 5′-endRACE products are combined to produce an intact full-length cDNA. Thismethod is described in Innis et al. (supra); and Frohman et al., Proc.Natl. Acad. Sci. USA 85:8998, 1988. Exemplary oligonucleotide primersuseful for amplifying RIM1 homolog sequences include, withoutlimitation:

A. GAATTAACCCTCACTAAAGGGAAARMGNGAYCAYATHAC (SEQ ID NO: 1); and B.GTAATACGACTCACTATAGGGTGYTTYTTNARRTCYTG (SEQ ID NO: 2).

For each of the above sequences, N is A, T, G or C.

Alternatively, any fungal cDNA or cDNA expression library may bescreened by functional complementation of an invasin mutant (forexample, the RIM1 and AFL1 mutants described herein) according tostandard methods described herein.

Confirmation of a sequence's relatedness to an invasin polypeptide maybe accomplished by a variety of conventional methods including, but notlimited to, functional complementation assays and sequence comparison ofthe homolog and its expressed product. In addition, the activity of thegene product may be evaluated according to any of the techniquesdescribed herein, for example, the functional or immunologicalproperties of its encoded product.

Once an invasin homolog is identified, it is cloned according tostandard methods and used for the construction of fungal expressionvectors according to standard methods.

Interactin, Polypeptides

The isolation of invasin protein sequences also facilitates theidentification of polypeptides which interact with the invasin proteins.Such polypeptide-encoding sequences are isolated by any standard twohybrid system (see, for example, Fields et al., Nature 1989,340:245-246; Yang et al., Science 1992, 257:680-682; Zervos et al., Cell1993, 72:223-232). For example, all or a part of the Tpk2 sequence maybe fused to a DNA binding domain (such as the GAL4 or LexA DNA bindingdomain). After establishing that this fusion protein does not itselfactivate expression of a reporter gene (for example, a lacZ or LEU2reporter gene) bearing appropriate DNA binding sites, this fusionprotein is used as an interaction target. Candidate interacting proteinsfused to an activation domain (for example, an acidic activation domain)are then co-expressed with the Tpk2 fusion in host cells, andinteracting proteins are identified by their ability to contact the Tpk2sequence and stimulate reporter gene expression. Tpk2-interactingproteins identified using this screening method provide good candidatesfor proteins that are involved in the acquired resistance signaltransduction pathway. An example of this method is described herein(infra).

Isolatin Fungal Homologs which Complement S. cerevisiae Mutations

One use of the S. cerevisiae mutant strains is to screen for homologs inother fungi which can complement the S. cerevisiae mutations. Here weprovide evidence for the efficacy of such an approach. We haveidentified the C. albicans homolog of INV8 (referred to as RIM1 (FIG.5A; SEQ ID NO: 3)). When the full length RIM1 was expressed from aplasmid in S. cerevisiae inv8 strains, the cells still showed an inv8phenotype. However, the homologs of RIM1 (S. cerevisiae INV8, Y.lipolytica RIM101, and A. nidulans pacC) require proteolytic processingfor activation. It is likely that the S. cerevisiae protease could notcleave C. albicans Rim1 (FIG. 5A; SEQ ID NO: 4). In support of this, aRIM1 gene truncated just prior to the protease cleavage site, was foundto complement the inv8 phenotype in S. cerevisiae (FIG. 5B.) This makesit likely that a Rim1 protease exists in C. albicans, and this proteasewould be considered to be a homolog of INV14. Based on our discovery ofthe correlation between S. cerevisiae invasion and secondary metaboliteproduction by other fungi, it is likely that many if not all of the S.cerevisiae INV genes will have homologs in diverse fungi. Indeed, INV5,INV9 and INV14 are predicted to encode proteins with high amino acidsimilarity to the products of the A. nidulans genes palA, palF, andpalB, respectively. We found that the published sequence for the genomicregion near INV9 contains several errors. The correct DNA sequence ofINV9 (SEQ ID NO: 5) and the sequence of the predicted polypeptide (SEQID NO: 6) is shown in FIG. 6A-B. INV5/palA, INV9/palF, and INV 14/palBare required to activate Inv8/PacC in their respective organisms. Thus,these genes are structurally and functionally conserved genes. Inaddition, homologs of lNV9, INV11, and INV13 appear on the list ofpartially sequenced C. albicans genes (http://alces.med.umn.edu). AnINV11 homolog in the fungus Kluveromyces lactis has also been partiallysequenced (GENBANK locus: KLAJ9848), as have homologs of INV1 (GENBANKlocus: SPBC3B9), INV13 (GENBANK locus: SPBC4B4) and INV15 (GENBANKlocus: SPAC1B3) in the fungus Schizosaccharomyces pombe. An STP22homolog has been identified in the fungus Saccharomyces carlsbergensis(GENBANK locus: SCZ86109). Of the 12 cloned genes in the INV pathway,six (INV5, INV8, and INV12-15) have obvious structural homologs outsidethe fungal kingdom. The existence of INV homologs in a wide variety offungi, including the pathogenic fungus, C. albicans, and one thatproduces the important secondary metabolite penicillin, A. nidulans,underscores the potential utility of manipulating these genes and/ortheir activities to affect both pathogenesis and secondary metaboliteproduction in diverse fungi. Additional homologs can be isolated byexpressing genes from fungal cDNA or genomic libraries in S. cerevisiaemutant strains and selecting for those transformants in which the mutantphenotype is complemented or enhanced.

Mutating Genes in Other Fungi to Determine Their Roles in Invasion

Following isolation of any specific homolog of a S. cerevisiae geneinvolved in invasion from any fungal species, we can determine the roleof that gene in pathogenesis or invasion by that fungus. Any such genethat activates or increases invasion is also likely to activate orincrease virulence. Deletion or inactivation of the function of thesegenes would likely reduce the virulence of that fungal strain. Thefollowing gene deletion example utilizes the C. albicans RIM1 gene fromthe previous section.

We tested whether the C. albicans RIM1 gene was required for invasivehyphal growth in C. albicans. A homozygous rim1/rim1 deletion wascreated. The C. albicans rim1/rim1 mutant does not form invasive hyphaein the presence of serum on agar at 37° C., while nonmutant C. albicansdoes (FIG. 7). The virulence of the mutated strains can be determinedusing a variety of animal models. One model of systemic infection isdescribed as an example. To test the relative virulence (infectivity) ofa mutant strain of Candida compared to a normal “wild-type” strain,mutant and normal Candida cells are injected into the tail veins ofseparate healthy mice. The mice are then observed for a period of twoweeks to one month. The relative numbers of mice killed by the mutantversus normal Candida provides an index of virulence (see Lo et al.,Cell 1997, 90: 939-949). Furthermore, any fungal gene that inhibits ordecreases invasion is also likely to inhibit or decrease secondarymetabolite production. Deleting or disrupting the function of any suchgene is likely to increase secondary metabolite production. Geneticengineering methods, including transformation and homologousrecombination techniques, are practiced in many fungi (Punt and van denHondel, Methods Enzymol. 1992, 216:447-457; Timberlake and Marshall,Science, 1989, 244:1313-1317; Fincham, Microbiol Rev. 1989, 53:148-170).Gene deletion techniques are currently practiced in many fungi,including, but not limited to, Candida albicans (Fonzi and Irwin,Genetics 1993, 134: 717-728), Ustilago maydis (Fotheringham and Hollman,Mol. Cell Biol. 1989, 9:4052-4055; Bolker et al., Mol. Gen. Genet. 1995,248:547-552), Yarrowia lipolytica (Neuveglise et al., Gene 1998,213:37-46; Chen et al., Appl. Microbiol. Biotechnol. 1997, 48:232-235;Cordero et al., Appl. Microbiol. Biotechnol. 1996, 46:143-148),Acremonium chrysogenum (Skatrud et al., Curr. Genet. 1987, 12:337-348;Walz and Kuck, Curr. Genet. 1993, 24:421-427), Magnaporthe grisea(Sweigard et al., Mol. Gen. Genet. 1992, 232:183-190); Kershaw et al.,EMBO J. 1998, 17:3838-3849), Histoplasma capsulatum (Woods et al., J.Bacteriol. 1998, 180:5135-5143) and Aspergillus sp. (Miller et al., Mol.Cell Biol. 1985, 5:1714-1721; de Ruiter-Jacobs et al., Curr. Genet.1989, 16:159-163; Gouka et al., Curr. Genet. 1995, 27:536-540; van denHombergh et al., Mol. Gen. Genet. 1996, 251:542-550; D'Enfert, Curr.Genet. 1996, 30:76-82; Weidner et al., Curr. Genet. 1998, 33:378-385).

This gene deletion technique allows for the selection of fungal homologswhich give the strongest phenotypes, as well as those which are invasionspecific. Some of the gene disruptions will likely produce nonpathogenicfungi. These genes and the encoded proteins are excellent candidates totarget with compounds. Other disruptions will likely producehyperinvasive fungi. The mutation of these genes in fungi such asAspergillus sp., Penicillium sp., Acremonium chrysogenum, Yarrowialipolytica, and Phaffia rhodozyma should lead to increased production ofsecondary metabolites of commercial value, such as beta-lactamantibiotics and their derivatives, or anti-hypercholesterolemic agentssuch as the statins.

A System to Increase Production of Secondary Metabolites

Fungi secrete secondary metabolites in order to produce an environmentfavorable for their survival. These metabolites have also been shown tohave a great number of commercial uses. We have discovered a correlationbetween secondary metabolite production and the regulation of invasion.The A. nidulans pacC gene, involved in regulation of the penicillinproduction, has a homolog in both S. cerevisiae and C. albicans whichhas a role in invasion. The A. nidulans genes palA, palD, and palF,involved in regulation of penicillin production, also have homologs inS. cerevisiae that are required for invasion. In addition, as describedabove, we have identified the homolog of pacC in C. albicans (RIM1) andshowed that it is also necessary for invasion. Similarly, many of thenutritional conditions that induce substrate invasion by S. cerevisiaealso lead to increased penicillin production in A. nidulans. Forexample, growth in the presence of limiting amounts of ammonia isimportant both for pseudohyphal growth in S. cerevisiae and productionof penicillin in A. nidulans (see Gimeno et al., supra, and Brakhage, A.A., Micro. Mol. Biol. Rev. 1998, 62: 547-585). Many of the genes we haveshown to regulate invasion, as well as expression from the FLO11promoter (infra), will also regulate production of secondary metabolitesin fungi such as Aspergillus sp., Penicillium sp., Acremoniumchrysogenum, Yarrowia lipolytica and Phaffia rhodozyma. This knowledgecan be used to produce fungal strains which produce greater amounts ofthese secondary metabolites, many of which are of great commercialvalue.

One example of how genetic manipulation can increase secondarymetabolite production utilizes the fungal invasion-inhibiting gene,HOG1. We have shown that HOG1 is a negative regulator of haploidinvasion and pseudohyphal growth in S. cerevisiae. Mutating or deletingHOG1 in S. cerevisiae produces a transgenic strain which invades itssubstrate in conditions in which a wild-type strain does not. Decreasingthe activity of the Hog1 protein in fungal species such as Aspergillussp., Penicillium sp, Acremonium chrysogenum, Yarrowia lipolytica andPhaffia rhodozyma, results in an increased production of secondarymetabolites through a de-repression of the signal transduction pathway.There are a variety of ways to decrease protein activity. One method isto delete the entire HOG1 gene or a portion thereof, using homologousrecombination techniques known to be applicable in fungi as describedherein. Similarly, mutagenesis of genes could also lead to decreasedprotein activity. Another method would be to apply a gene or compoundwhich, directly or indirectly, inhibits the expression of HOG1 or theactivity of Hog1. These genes and compounds can be isolated usingscreening techniques described herein. The methods described for HOG1are equally applicable to any fungal anti-invasion gene, including, butnot limited to, GRR1, IRA1, BEM2, TPK3, SFL1, SSD1, RIM15, CDC55, SWI4,and ELM1.

A second mechanism to increase production is to increase the activity ofa fungal invasion-promoting gene, such as homologs of INV8 or any otherfungal invasion-promoting gene. One method is to express such a genefrom a constitutively active promoter. Expression systems, such as thosewhich drive expression of genes from promoter sequence from either theAspergillus nidulans gpdA gene, the Acremonium chrysogenum ipnS gene, orthe Penicillium chrysogenum phoA gene, are well-known to those skilledin the art (Skatrud et al., Curr. Genet. 1987, 12:337-348; Kolar et al.,Gene 1988, 62:127-134; de Ruiter-Jacobs et al., Curr. Genet. 1989,16:159-163; Smith et al., Gene 1988, 114:211-216; Graessle et al., Appl.Environ. Microbiol. 1997, 63:753-756).

Another method to increase activity of a fungal invasion-promoting geneis to genetically alter the fungal invasion gene, either through theintroduction of an activating mutation or by creating fusions to othergenes that result in increased activity. The fusion proteins arepreferably constitutively active. A third method is to mutate the fungalinvasion gene such that the activity of the encoded protein isincreased. For example, S. cerevisiae Inv8 and its homolog, PacC,require proteolyic cleavage for transcriptional activating properties.We have shown that this cleavage is required for the nuclearlocalization of Inv8. Inability to truncate Inv8 results in itsaccumulation in the cytoplasm (FIG. 8). These data suggest thatactivation of Inv8 and its homologs is likely to be a consequence ofallowing the protein to gain access to its target genes in the nucleus.Thus, truncation of Inv8 or PacC caused by the introduction of a stopcodon upstream of the coding sequence for the protease cleavage sitewould create a constitutively active transcription factor. Anothermethod is to apply a compound which, directly or indirectly, increasesthe expression of INV8 or the activity of Inv8. These compounds can beisolated using screening techniques described herein. S. cerevisiae INV8is homologous to Y. lipolytica RIM101, and pacC from A. nidulans, A.niger, and P. chrysogenum. pacC is known to directly regulate penicillinproduction; penicillin production is regulated by the activated form ofthe transcription factor. Thus, increased PacC activity using any of thedescribed methods will lead to increased penicillin production.

The FLO11 Promoter is a Reporter of Fungal Invasion

The cell surface protein Flo11 has been reported to be required forinvasive and filamentous growth (Lambrechts et al., 1996; Liu et al.,1996; Lo and Dranganis, 1996; Lo and Dranganis, 1998; Flo11 is referredto as Muc1 in S. cerevisiae var. diastaticus). The FLO genes encodeproteins required for cell-cell adhesion (Teunissen and Steensma, 1995).The MAP kinase pathway, involving four protein kinases (Ste20, Ste11,Ste7, and Kss1), regulates the activity of the transcription factorSte12/Tec1. One of the genes regulated by this signal transductioncascade is FLO11.

We have discovered that FLO11 is a downstream target for other signaltransduction pathways that regulate filamentous growth in diploids andinvasion in haploids. While it was known that one pathway whichregulated these phenotypes also regulated FLO11 expression, it wassurprising that FLO11 appeared to be under such complex regulation byseveral signal transduction pathways. These findings are outlined below.

An example of another signal transduction cascade which regulates FLO11expression is the cAMP/PKA pathway. The evidence which implicated FLO11as a target of the cAMP pathway follows. Since both the MAPK pathway andthe cAMP/PKA pathway are activated for filamentation by the sameactivator, Ras2, analysis of the distinct role of cAMP on filamentousgrowth requires the ability to activate the PKA branch independently ofRas. To achieve this goal, we constructed a strain (ras1 ras2 pde2)lacking both RAS genes (RAS1 and RAS2) and the high-affinity cAMPphosphodiesterase, PDE2. Since Ras1 and Ras2 are required for theactivation of adenylate cyclase, Cyr1 and PDE2 encodes thephosphodiesterase required for cAMP hydrolysis, a ras1 ras2 pde2 strainis impaired in the synthesis and breakdown of cAMP. Such a strain shouldbe dependent upon exogenous cAMP for induction of the A kinase and, asno Ras-induced signal can be transmitted to the MAPK cascade, theeffects of cAMP on filamentation should be independent of the Ras/MAPKsignal.

The ras1 ras2 pde2 strain (SR957) requires cAMP for growth on YPD (yeastextract, peptone, dextrose), but grows without cAMP on SC (syntheticcomplete), SLAD (synthetic low ammonia dextrose), and YNB (yeastnitrogen base), media where it displays hyper-accumulation of glycogen,indicative of low cAMP levels. We presume that the ability of thistriple mutant to grow without added cAMP, as has been observed by others(Nikawa et al., 1987), results from basal cyclase activity that issufficient to provide internal cAMP. In support of this notion, weobserved that growth of our ras1 ras2 pde2 strain depended upon afunctional cyclase (CYR1) gene.

A diploid ras1 ras2 pde2 (SR959) strain grows on SLAD medium withoutcAMP but does not form pseudohyphae. However, on SLAD medium containingcAMP, the strain is extremely filamentous. Moreover, the addition ofcAMP leads not only to induction of filamentation, but also to invasionof the substrate. In the presence of cAMP, the ras1 ras2pde2 strain(SR959) is invasive on all media tested (YPD, SC, SLAD). Since invasivegrowth is usually observed with haploid strains on YPD, both the celltype signal and the nutritional signal can be bypassed by high cAMPlevels in the cell.

To determine whether cAMP induces filamentation by activating the MAPKpathway, we measured the expression of the Kss1 MAPK pathway-specificreporter FG::Ty1-lacZ (Mosch et al., supra) in ras1 ras2,pde2 strainsgrown on SLAD plates containing concentrations of cAMP that inducefilamentation. The level of expression of the FG::Ty1-lacZ reporter inthe ras1 ras2 pde2 strain (SR959) was not altered by these cAMP levels.Moreover, cAMP induced filamentation in a ste12 ras1 ras2 pde2 deletionstrain (SR1088), indicating that the cAMP/PKA pathway acts in parallelwith the MAPK pathway.

We demonstrated the induction of FLO11 mRNA by cAMP in the ras1 ras2pde2 strain, but not in a wild-type background. The FLO11 transcript wasundetectable when the triple mutant strain is grown without cAMP andstrongly induced in the presence of 2 mM cAMP. Expression of FLO1, whichencodes Flo1, another cell surface protein required for flocculation and26% identical to Flo11, is only induced 1.4 fold under identicalconditions. This result shows that the strong induction of FLO11 is nota general feature of all flocculation genes. The correspondence betweencAMP induction of FLO11 and the morphological changes observed when thecells are grown in the presence of cAMP is further supported by thephenotype of the FLO11 deletion; in the flo11 ras1 ras2pde2 strain(SR1121), cAMP fails to induce either invasion or filamentation. Thesedata suggest that FLO11 is a key target of a cAMP-dependent signalingpathway, one that is required for the induction of invasive andfilamentous growth.

The enhanced FLO11 transcription in the presence of cAMP is correlatedwith a change in cellular morphology. Cells grown in liquid SC with 2 mMcAMP show pseudohyphal-like chains of cells, whereas the majority ofcells grown in liquid SC without CAMP are either single cells or cellswith a single bud. The effect of cAMP on cell-cell attachment is muchmore pronounced than the effect of the cyclic nucleotide on cellelongation.

The use of the FLO11 promoter as a reporter of invasion and filamentousgrowth appears to be universal. As shown in FIG. 9, the FLO11 promoteris an excellent reporter for invasion and pseudohyphal growth. Formutants that reduce invasion or pseudohyphal growth, there was a directcorrelation between FLO11 expression and invasion/pseudohyphal growthfor afl1, ste12, tec1, flo8, inv8, ras2, tpk2, phd1, phd2, inv6, inv7,whi3, dhh1, mep2, ptc1, mcm1, inv1, inv13, and inv14. In addition, hog1,grr1, ira1, tpk3, and sfl1 mutants, which exhibited increasedinvasion/pseudohyphal growth, also displayed increased FLO11 expression.For example, FLO11 is transcriptionally regulated by both Tpk2 and Sfl1.tpk2 mutants showed a 10-fold reduction in FLO11 expression, whereastpk3 mutants had a threefold elevation. The levels of FLO11 mRNA werealso reduced 10-fold in tpk2 tpk3 double mutants. FLO11 mRNA levels werealso increased threefold in an sfl1 mutant and were similarly increasedin an sfl1 tpk2 double mutant. The levels of FLO11 mRNA in the sfl1 tpk3double mutant exceeded that of either single mutant.

Not only is FLO11 an excellent reporter of pseudohyphal growth andinvasion, it is also central to invasion and pseudohyphal growth. sfl1flo11 haploid mutant strains were defective for invasive growth on richmedium, and sfl1 flo11 homozygous diploid mutants were defective forpseudohyphal development. Thus, flo11 loss of function blocked thehyperinvasive and hyperfilamentous phenotypes of sfl1 loss of function.However, the defect of the sfl1 flo11 double mutant was not as severe asthat of the flo11 single mutant, suggesting that FLO11 is not the onlydownstream target of Sfl1 involved in pseudohyphal development andhaploid invasion.

It is reasonable to assume that many other genes that have been placedin signal transduction pathways, including the genes shown in FIG. 10,will also be required for FLO11 expression. Based on these results, andour recent identification of a number of additional genes that regulateinvasion (and quite likely FLO11 expression), we have constructed awiring diagram for the genetic circuitry that regulates FLO11 (FIG. 10).These genes, listed in Table 1, all represent candidate targets fornovel antifungal agents that act by blocking invasion and/or dimorphicgrowth.

To determine how much of this 5′ region of FLO11 was required for theregulated expression of FLO11, we examined the expression ofplasmid-based FLO11::lacZ reporter constructs containing deletions inthis non-coding region. Fourteen serial 200 bp deletions wereconstructed that span the region 2800 bases (bp −2800) upstream from theFLO11 initiation codon. A FLO11-lacZ reporter plasmid was constructed byamplifying the 3 kb region 5′ of the ATG by PCR using primers containinga BglII site at the end and cloning into YEp355. (Sequences of primersused are 5′-CGCACACTATGCAAAGACCGAGATCTTCC-3′ (SEQ ID NO: 7) and5′-GAAGATCTTCTCCACATACCAATCACTCG-3′) (SEQ ID NO: 8). The 14 individualdeletions in this reporter were constructed by primer overlap methodusing a KS+-Bluescript plasmid containing the 3 kb FLO11-promoter region(subcloned from YEp355-FLO11::lacZ as EcoRI/HindIII fragment). Aftermutagenesis the partially deleted flo11-nn promoter sequence wasrecloned into YEp355. The primers used for this purpose were:

-3′ #1F 5′-CAAGCATTTACGTTACTGCGAAAATCCATATACGCACACT-3′ (SEQ ID NO: 9)#1R 5′-AGTGTGCGTATATGGATTTTCGCAGTAACGTAAATGCTTG-3′ (SEQ ID NO: 10) #2F5′-TGATGAGGTAACCTTTACAACTCTCTTCTAGTTCAAGAAC-3′ (SEQ ID NO: 11) #2R5′-GTTCTTGAACTAGAAGAGAGTTGTAAAGGTTACCTCATCA-3′ (SEQ ID NO: 12) #3F5′-TTTCAATTCAATGGATTTGGAATGTCATAGAGTTACCAAT-3′ (SEQ ID NO: 13 #3R5′-ATTGGTAACTCTATGACATTCCAAATCCATTGAATTGAAA-3′ (SEQ ID NO: 14) #4F5′-ATTTCTGCCTATACTCTTAAAGGTATTCGTTTGTTTACTA-3′ (SEQ ID NO: 15) #4R5′-TAGTAAACAAACGAATACCTTTAAGAGTATAGGCAGAAAT-3′ (SEQ ID NO: 16) #5F5′-TTGGGGCTAAGAATGGACACAGATCAGTCATTCATGTTGT-3′ (SEQ ID NO: 17) #5R5′-ACAACATGAATGACTGATCTGTGTCCATTCTTAGCCCCAA-3′ (SEQ ID NO: 18) #6F5′-GGGTGTGCCTGGAAAGTTCATTCCCTTTTCTTTTTCTTTG-3′ (SEQ ID NO: 19) #6R5′-CAAAGAAAAAGAAAAGGGAATGAACTTTCCAGGCACACCC-3′ (SEQ ID NO: 20) #7F5′-CAAAACTTTAGGAATACCGGAAATTAAGGTTTTTTTCTTC-3′ (SEQ ID NO: 21) #7R5′-GAAGAAAAAAACCTTAATTTCCGGTATTCCTAAAGTTTTG-3′ (SEQ ID NO: 22) #8F5′-CGAATGTGAATGCGCTAATCTTGTGTGCCTACGCCAGCCC-3′ (SEQ ID NO: 23) #8R5′-GGGCTGGCGTAGGCACACAAGATTAGCGCATTCACATTCG-3′ (SEQ ID NO: 24) #9F5′-AGACAAAAAATAGGAAAAGTGGTATTTCCACCACATGAAA-3′ (SEQ ID NO: 25) #9R5′-TTTCATGTGGTGGAAATACCACTTTTCCTATTTTTTGTCT-3′ (SEQ ID NO: 26) #10F5′-TTAGTGCGGAATACTTTCCTTTAATTAGTGATGGTTCTCA-3′ (SEQ ID NO: 27) #10R5′-TGAGAACCATCACTAATTAAAGGAAAGTATTCCGCACTAA-3′ (SEQ ID NO: 28) #11F5′-CAGTGCTTTCAACACCTTTTATTCTCATCGAGAGCCGAGC-3′ (SEQ ID NO: 29) #11R5′-GCTCGGCTCTCGATGAGAATAAAAGGTGTTGAAAGCACTG-3′ (SEQ ID NO: 30) #12F5′-GTAGCTGAAAAGTCCATCTACATCTGTGTGCCATGTCAGA-3′ (SEQ ID NO: 31) #12R5′-TCTGACATGGCACACAGATGTAGATGGACTTTTCAGCTAC-3′ (SEQ ID NO: 32) #13F5′-GAGATTATCTTGGGATCTATTCGAATTATGAATGATACTA-3′ (SEQ ID NO: 33) #13R5′-TAGTATCATTCATAATTCGAATAGATCCCAAGATAATCTC-3′ (SEQ ID NO: 34) #14F5′-GTTTTGGCTCAATGGGACCGTTCACAAATTTACGGCTAAT-3′ (SEQ ID NO: 35) #14R5′-ATTAGCCGTAAATTTGTGAACGGTCCCATTGAGCCAAAAC-3′ (SEQ ID NO: 36)

The resulting 14 plasmids for the deletion series were transformed intostrain 10560-2B. At least three independent clones were tested usingfilter assays for equivalent expression of β-galactosidase. Diploidstrains were created by mating of the respective 10560-2B flo11-nntransformant with strain 10560-SB by selection on YNB containing onlyleucine as a supplement.

The 14 plasmids containing the 400 bp sequence elements were transformedinto 10560-2B, L5795, L5816, L6149, L6150 and SR957. At least threeindependent clones were tested using filter assays for equivalentexpression of β-galactosidase. Filter assays were used to test theeffect of α-factor (5 μM).

The large number of FLO11 promoter elements and multiple conditions thataffect those elements required some economy in the experimental design.The measurement of β-galactosidase from our plasmid-based reportercontaining each of these elements permitted the rapid construction ofstrains and reproducible measurement of reporter activity. We confinedthe analysis to selective media so that the strains retained theplasmids. Our β-galactosidase assays show in general a higher activityfor haploid strains than for diploid strains. For rich medium thisresult is in agreement with that found previously when FLO11 expressionwas monitored by measurement of steady state mRNA-levels. When wemeasure FLO11::lacZ after 24-26 h (post diauxic growth) or on SLADmedium, haploid cells show an induction of more than 10 fold and diploidcells, 5 fold induction. This large induction of FLO11::lacZ for haploidcells on SLAD was not observed in the experiments where FLO11 mRNA wasmeasured. This discrepancy could be due either to a difference in thestability of mRNA vs. protein or the differences in the times at whichthe cultures were sampled. The expression of FLO11 as measured by eithermethod is extremely sensitive to growth conditions and growth phase.

Total RNA was prepared using hot acid phenol and northern blots wereperformed as described in Ausubel et al., (supra). Strains deleted forras1 ras2 pde2 were pre-grown in SC+1 mM cAMP to OD600=1.0. The cellswere washed twice using SC and diluted to an OD600=0.3 into fresh SCmedium or SC medium+2 mM cAMP. The cells were grown to OD600=1.0 andharvested. 10-15 μg of RNA were separated on a formaldehyde containingagarose gel. For hybridization to FLO11 mRNA, a probe corresponding tobp 3502-4093 of the FLO11 ORF was used.

Cells for β-galactosidase assays of FG::TyA-lacZ were incubated onSLAD-plates for three days; cells for FLO11::lacZ expression studieswere grown in the respective liquid media and quantitated according toMosch et al. (supra).

Cells for quantitation of FLO11::lacZ expression in exponential growthphase were inoculated from confluent 20 h grown cultures 1:20 in freshmedium and grown for 4-6 h. Cells for quantitation of FLO11::lacZexpression after the post-diauxic shift were grown for 24-26 h. The samecultures were used to inoculate the exponentially growing cultures.Cells for quantitation of FLO11::lacZ expression in SLAD-medium werepre-grown for 20 h in SC-medium, washed twice with 2% glucose, diluted1:5 into SLAD-medium and grown for 4-6 hours. For detection of cAMPinduced FLO11 promoter segments strain SR957 harboring the individualplasmids was grown overnight in selective medium containing 1 mM cAMP,transferred to media containing no cAMP for 10 h and split into 2cultures containing 2 mM cAMP or no cAMP and grown for 4 h beforeharvesting.

Expression of the deletions was assayed in haploid and diploid strains.Since FLO11 expression varies with growth phase of the cells, weanalyzed exponentially growing cells on SC, cells grown on SC until theglucose had been depleted (post-diauxic), and cells on SLAD, a mediumthat is high in glucose and low in nitrogen.

Enzymatic assays on the individual FLO11::lacZ promoter deletionsreveals an unusually long promoter with many sites. In subsequentsections, a site is tentatively assigned as an upstream repression site(URS) if its deletion leads to at least 3-fold enhanced expression oflacZ, and as an upstream activation site (UAS) if its deletion leads toat least 30% reduced expression.

Analysis of cis-acting elements by enzymatic assays of the individualflo11-lacZ promoter deletions reveals that the intact FLO11 promoter ishighly repressed. The deletions define at least nine URS elements whoseactivity depends on state of growth, nutrient conditions, and cell type.One of the URS elements is defined by flo11-14 (bp-2600-2800), showingthat cis-acting elements are present at least 2800 bases from theputative FLO11 coding region. In general, haploid strains show strongerrepression than diploid strains.

A clear way to visualize the activity of the sites of repression is bycomparison of the lacZ activity for each of the conditions with that ofa haploid grown on SLAD. This comparison reveals URS elements withinflo11-4,-5,-7,-8,-12,-13, and flo11-14. A subset of these elements iskey to repression on all media, but the strongest effect is in haploidsgrown on SLAD. Haploid-specific effects are found for deletionsflo11-4,-10,-11,-12, and flo11-13. Clearly, there are sites inflo11-4,flo11-12, and flo11-13 that function in the haploid-specificnitrogen repression of FLO11.

There are other differences between haploid and diploid strains, themost notable of which being: 1) in diploids on SC (exponential growth),flo11-4 has a 2-fold reduced expression level, whereas in haploids ithas 33-fold elevated expression; 2) in diploids after the diauxic shift,flo11-11 has a 3-fold reduced level whereas in haploids it has 12-foldelevated expression. In diploid cells, flo11-4,flo11-10, and flo11-11act as UAS elements. flo11-5 is a strongly nitrogen-regulated site inboth haploids and diploids.

Deletion analysis also revealed sequence elements required forexpression of FLO11. Under all conditions tested, flo11-6 had a dramaticreduction in expression, suggesting that the sequence deleted in flo11-6(−1200 to −1000) contained a strong UAS. Furthermore, flo11-1,flo11-2,and flo11-3 show consistently lower activity, as compared to thewild-type FLO11-lacZ reporter construct, identifying these as UASelements in the FLO11 promoter. The reduced expression of flo11-1 islikely to be a consequence of the deletion of the TATAA region in thisconstruct.

We made two flo11 promoter mutations in the chromosome at the FLO11locus. In one, flo11-16, we deleted virtually the entire promoter region(from −150 to −2947). In the other, we replaced the entire wild-typepromoter with flo11-6. Chromosomal deletion of the FLO11 promoter wasdone by replacing base pairs −2947 to −150 with URA3 (sequences ofprimers used:

5′-ACCACAACATGACGAGGGATAATAACTGATGAATAGGGTGCTTTTTATACTCTGTGCGGTATTTCACACC-3′ (SEQ ID NO: 37) and5′-TAAGGAGTCGTACCGCCAACTAAATCTGAATAACAATTTGGCTGCTAGAAGCAGATTGTACTGAGAGTGC-3′; SEQ ID NO: 38), resulting in flo11-16 (SR 1172)To generate flo11-6 in the chromosomal FLO11 promoter region, the URA3gene was replaced by transformation with EcoRI/HindIII digestedKS+flo11-D6 and selection on 5-FOA to yield deletion of base pairs −1000to −1200 (SR 1174).

A strain carrying either flo11-16 or flo11-6 is completely defective inhaploid invasive growth and, as diploids (e.g.,flo11-6flo11-6), showseverely reduced filamentation. The haploid invasion defect is as severeas that of a deletion of the FLO11 coding region. The results withflo11-6 support our conclusions based on the data from the lacZ plasmidconstructs, which identified the segment deleted in the flo11-6construct as a critical UAS for FLO11 expression.

In a second approach to identify UAS elements of the FLO11 promoter, wedesigned 14 individual sequence elements of approximately 400 basepairs, to test activation of a lacZ reporter fused to a basaltranscriptional unit (FIG. 11). To determine UAS-sequence elements, 14individual 400 bp elements, overlapping by 200 bp were amplified by PCRand cloned into pLG669Z. This vector contains one codon of the CYC1 genefused to the lacZ gene, and the fused gene is preceded by 1100nucleotides that lie upstream from CYC1. The promoter fragment has XhoIrestriction sites at positions −683 and −249. The XhoI fragment has beenexcised and replaced with a fragment from the FLO11 promoter. Thefragments correspond to the following positions relative to the startcodon of FLO11: −1 to −421 (FLO11-2/1), −181 to −618 (FLO11-3/2), −380to −819 (FLO11-4/3), −580 to −1017 (FLO11-5/4), −778 to−1219(FLO11-6/5), −980 to −1420 (FLO11-7/6), −1181 to −1619 (FLO11-8/7),−1380 to −1819 (FLO11-9/8), −1572 to −2019 (FLO11-10/9), −1780to −2219(FLO11-11/10), −1980 to −2419 (FLO11-12/11), −2180 to −2619(FLO11-13/12), −2380 to −2819 (FLO11-14/13), −2580 to −2983(FLO11-15/14). For cloning purposes, a restriction site (XhoI) wasintroduced at the 5′ end of the PCR-primers. The primers used for thispurpose were:

#1R 5′-CCGCTCGAGAGTGTGCGTATATGGATTTT-3′ (SEQ ID NO: 39) #2F5′-CCGCTCGAGTGATGAGGTAACCTTTACAA-3′ (SEQ ID NO: 40) #2R5′-CCGCTCGAGGTTCTTGAACTAGAAGAGAG-3′ (SEQ ID NO: 41) #3F5′-CCGCTCGAGTTTCAATTCAATGGATTTGG-3′ (SEQ ID NO: 42) #3R5′-CCGCTCGAGATTGGTAACTCTATGACATT-3′ (SEQ ID NO: 43) #4F5′-CCGCTCGAGATTTCTGCCTATACTCTTAA-3′ (SEQ ID NO: 44) #4R5′-CCGCTCGAGTAGTAAACAAACGAATACCT-3′ (SEQ ID NO: 45) #5F5′-CCGCTCGAGTTGGGGCTAAGAATGGACTT-3′ (SEQ ID NO: 46) #5R5′-CCGCTCGAGACAACATGAATGACTGATCT-3′ (SEQ ID NO: 47) #6F5′-CCGCTCGAGGGGTGTGCCTGGAAAGTTCA-3′ (SEQ ID NO: 48) #6R5′-CCGCTCGAGCAAAGAAAAAGAAAAGGGAA-3′ (SEQ ID NO: 49) #7F5′-CCGCTCGAGCAAAACTTTAGGAATACCGG-3′ (SEQ ID NO: 50) #7R5′-CCGCTCGAGGAAGAAAAAAACCTTAATTT-3′ (SEQ ID NO: 51) #8F5′-CCGCTCGAGCGAATGTGAATGCGCTAATC-3′ (SEQ ID NO: 52) #8R5′-CCGCTCGAGGGGCTGGCGTAGGCACACAA-3′ (SEQ ID NO: 53) #9F5′-CCGCTCGAGAGACAAAAAATAGGAAAAGT-3′ (SEQ ID NO: 54) #9R5′-CCGCTCGAGGTAGCAGGTTTCATGTGGTG-3′ (SEQ ID NO: 55) #10F5′-CCGCTCGAGTTAGTGCGGAATACTTTCCT-3′ (SEQ ID NO: 56) #10R5′-CCGCTCGAGTGAGAACCATCACTAATTAA-3′ (SEQ ID NO: 57) #11F5′-CCGCTCGAGCAGTGCTTTCAACACCTTTT-3′ (SEQ ID NO: 58) #11R5′-CCGCTCGAGGCTCGGCTCTCGATGAGAAT-3′ (SEQ ID NO: 59) #12F5′-CCGCTCGAGGTAGCTGAAAAGTCCATCTA-3′ (SEQ ID NO: 60) #12R5′-CCGCTCGAGTCTGACATGGCACACAGATG-3′ (SEQ ID NO: 61) #13F5′-CCGCTCGAGGAGATTATCTTGGGATCTAT-3′ (SEQ ID NO: 62) #13R5′-CCGCTCGAGTAGTATCATTCATAATTCGA-3′ (SEQ ID NO: 63) #14F5′-CCGCTCGAGGTTTTGGCTCAATGGGACCG-3′ (SEQ ID NO: 64) #14R5′-CCGCTCGAGATTAGCCGTAAATTTGTGAA-3′ (SEQ ID NO: 65) #15F5′-CCGCTCGAGCTCCACATACCAATCACTCG-3′ (SEQ ID NO: 66) #15R5′-CCGCTCGAGTAGTTAAACGTTTTATTAGC-3′ (SEQ ID NO: 67)

This reporter construct series identified UAS elements in the segmentsFLO11-2/1 and FLO11-3/2 that overlap between bp −200 and −400, FLO11-6/5and FLO11-7/6 that overlap between bp −1200 and −1000, as well asFLO11-10/9 and FLO11-11/10 that overlap between bp −2000 and −1800.These sequence elements show a more than 2-fold increase inβ-galactosidase activity as compared to the reporter plasmid containingthe basal transcriptional unit but without any insert. The activity ofFLO11-2/1, FLO11-3/2 and FLO11-11/10 are induced in post-diauxic cells,leading to an induction of up to 200-fold. These results suggested thatelements FLO11-2, FLO11-3, and FLO11-11 are required for FLO11expression in later stages of growth. Taken together with the deletionanalysis, these data suggested that there were at least four UASelements in the FLO11 promoter. The FLO11 promoter has extensivehomologies to the promoter regions of the MUC1, STA1, STA2, and STA3genes from S. cerevisiae var. diastaticus.

The types of promoter analyses described above, in combination with theseries of mutant strains identified which have defects in diploidfilamentous growth or haploid invasion, allows for the identification oftrans-acting elements and the regions of the FLO11 promoter that aretargeted by them. In one example, we transformed the 14 individual 400bp sequence elements into strains deleted for FLO8, STE12, TEC1, or forboth STE12 and TEC1.

Deletion of FLO8 led to a severe reduction in the expression ofFLO11-6/5 and FLO11-7/6 in both exponential (8-fold and 4-foldreduction, respectively) and post-diauxic growth (5-fold for bothelements). These elements are also induced by high cAMP levels. FLO8function is also required for maximum expression of FLO11-3/2 andFLO11-8/7 in post-diauxic growth phase, but not in exponential growth, aresult suggesting that Flo8 may function differently depending uponnutrient conditions.

Deletion of STE12 had a stronger effect in exponentially-growing cellsthan in post-diauxic cells. In exponentially-growing cells lackingSte12, the FLO11-6/5, FLO11-10/9, and FLO11-11/10 segments showed atleast a 3-fold reduction in expression. In post-diauxic cells, onlyFLO11-10/9 showed reduced expression in an STE12 deletion strain.

Deletion of TEC1 had significant effects on expression of the FLO11insertion reporter series, but the reduction could be observed only inexponentially-growing cells and was less severe than that observed forstrains deleted for STE12 or FLO8. This observation agrees with thenorthern analysis of the intact FLO11 promoter, where FLO11 mRNA levelsshowed less of a reduction in a strain deleted for TEC1 (L6149) than itdid in strains deleted for STE12 (L5795) or FLO8 (L5816). FLO11-6/5,FLO11-10/9, and FLO11-12/11 were dependent upon TEC1 inexponentially-growing cells. Deletion of TEC1 in a ste12 strain showed amodest reduction in the expression of FLO11-10/9 as compared to thesingle mutant strains. However, since the deletion of STE12 led toa >10-fold stronger effect than deletion of TEC1, this result mayindicate a Tec1-independent role for Ste12 at this site.

Our results suggest that Flo8 and Ste12/Tec1 act via multiple sites inthe FLO11 promoter that are largely non-overlapping. The strongesteffect of Flo8 and Ste12, both in exponential and post-diauxic cells,was observed with two distinct sequence elements of the FLO11 promoter.Flo8 acted on the sequence element defined by flo11-6 (bp −1200 to−1000),whereas Ste12 acted on FLO11-10/9 (bp −1800 to −1600).FLO11-12/11 and FLO11-10/9 were targeted both by Tec1 and Ste12. Theexistence of spatially distinct sites for different transcriptionfactors provides strong evidence in support for the combinatorialcontrol over FLO11 transcription.

In a second example, wve measured the expression of lacZ from fragmentsof the FLO11 promoter in strains mutant for AFL1. The diploids of thismutant strain do not forn filaments or invade under conditions wherewild-type yeast do. We discovered that one element, FLO11-11/10, wascompletely dependent upon AFL1 for expression. We also found thatexpression from FLO11 promoter fragment 11/10 was induced more that15-fold as yeast cells change from log phase growth to stationary phasegrowth. Furthermore, this stationary phase induction of transcriptionfrom FLO11-11/10 was completely dependent on AFL1.

As shown above, FLO11 expression is dependent upon numerous genesinvolved in pseudohyphal growth and haploid invasion. Thus, by combiningthe analysis of the FLO11 promoter with the variety of mutant strains,we will be able to map the trans-acting elements which regulate FLO11,as well as the important promoter elements which serve as targets. Asmany of the S. cerevisiae genes have homologs in other fungi, thisanalysis will allow for identification of putative targets for compoundswhich prevent fungal invasion or which promote production of secondarymetabolites.

In another example of using FLO11 promoter fragments and deletions incombination with mutant strains, the sites within the promoter where thecAMP signal activates FLO11 transcription were determined. Wetransformed the 400 bp reporter series into a ras1 ras2pde2 mutantstrain (SR957), a strain where internal cAMP levels can be manipulatedby adding cAMP to the media. Addition of 2 mM cAMP to SR957 led to a3-fold induction of FLO11-6/5 and a 2-fold induction of FLO11-7/6,FLO11-8/7 and FLO11-10/9, as compared to SR957 grown without cAMP. Theseresults suggest that trans-acting elements upregulate FLO11 expressionthrough a cAMP-mediated signal via more than one cis-acting element. Thesegment most strongly induced by cAMP is defined by the overlappingelements FLO11-6/5 and FLO11-7/6 (bp −1200 to −1000), the same elementtargeted by Flo8. As shown earlier, this element is also required forinduction of invasive and filamentous growth.

As described above, FLO11 transcripts were not detectable in a strain(L5816, flo8-2) that contains a deletion of the FLO8 gene. Furthermore,FLO11 induction by cAMP is blocked in a ras1 ras2pde2 strain (SR108I)carrying a deletion of FLO8. The ste12 ras1 ras2pde2 strain (SR1088),like the flo8 ras1 ras2pde2 strain (SR1081), has dramatically reducedexpression of FLO11. However, FLO11 transcription can be induced by cAMPin the ste12 ras1 ras2pde2 strain. The induction of FLO11 by cAMP in theste12 mutant and not in the flo8 mutant is consistent with thephenotypes of the corresponding strains: a flo8 ras1 ras2 pde2 strain isunable to form filaments on SLAD plates or to invade the agar on YPDplates, even in the presence of cAMP, whereas ste12 ras1 ras2pde2 isboth invasive and able to form filaments on YPD or SLAD platescontaining 2 mM cAMP. Thus, high cAMP levels can bypass the requirementfor the MAPK cascade transcription factor Ste12, but not the requirementfor Flo8 in the activation of FLO11 transcription. These experimentssupport a model in which several signal transduction cascades convergeon the promoter of FLO11.

The suppression patterns of ste12 and flo8 mutants by overexpression ofFLO8 and STE12, respectively, supports a model of theirjoint controlover FLO11. Overexpression of FLO8 in a ste12 strain (SR1021) andoverexpression of STE12 in a flo8 strain (SR 1134) suppressed thepseudohyphal and invasion defects of the mutants. The morphologies ofthe pseudohyphae in the “suppressed” strains were not identical to thatof wild-type. In flo8 strains overexpressing STE12, the cells of eachpseudohyphal strand appear more elongate than the cells of wild-typepseudohyphae. In ste12 strains overexpressing FLO8, the cells are notlonger than wild type. However, they have a denser network of filaments.This colony morphology is similar to that of strains that are induced toform filaments by cAMP (SR959).

The patterns of suppression by overexpression were reflected in theFLO11 expression pattern. Overexpression of FLO8 enhanced the expressionof FLO11 10-fold in a ste12 mutant (SR1021). The reciprocal experimentin which STE12 was overexpressed is more difficult because high levelsof Ste12 are toxic. To control the levels of STE12, we used a GAL::STE12construct which could be regulated by galactose. In a flo8 strain (SR1134) containing this GAL::STE12 construct on a plasmid, the FLO11transcript levels were increased 3-fold on SC-glucose medium as comparedto the strain transformed with the control plasmid (SR1097). Thisincrease probably represented incomplete repression of the GAL promoter.If STE12 was induced by incubation for four hours in SC-galactosemedium, FLO11 expression was increased 10-fold.

To determine whether a high level of internal cAMP stimulated FLO11transcription, we deleted IRA1 in an otherwise wild-type strain. Ira1 isa Ras-GAP that inactivates Ras-GTP by converting it to Ras-GDP. Loss ofIra1 function leads to a higher proportion of activated Ras and thus toelevated cAMP levels in the cell (Tanaka et al., 1989). In the ira1mutant strain (SR599), FLO11 transcripts were strongly induced. ThisFLO11 induction was reflected in the hyperinvasive phenotype of strainsdevoid of IRA1 function. A ste12 Ira1 mutant (SR 1133) was stillhyperinvasive, illustrating that at least some of the cAMP signal wasindependent of the MAPK pathway. However, flo11 ira1 (SR1079) or flo8ira1 (SR 1132) strains failed to invade. These results are consistentwith the interpretation that Flo8 is required for the induction of FLO11by high internal and external cAMP levels. A strain (SR 1132) lackingboth FLO8 and IRA1 had dramatically reduced levels of FLO11 transcripts,whereas deletion of STE12 in an ira1 background (SR 1133) still showedFLO11 transcript levels comparable to wild type. These results areconsistent with the hyper-invasive phenotype of the ira1 ste12 strainand the non-invasive phenotype of the ira1 flo8 strain.

Analysis of the regulation of the FLO11 promoter in S. cerevisiae hasprovided, and is likely to continue to provide, valuable informationregarding the regulation of invasion and secondary metabolite productionin diverse fungal species. Promoters of other genes involved in invasionor secondary metabolite production from S. cerevisiae or other fungalspecies are similarly likely to provide further valuable informationabout the regulation of invasion and secondary metabolite production. Anexample of such a promoter is the ECE1 gene promoter from Candidaalbicans. Expression of the Candida albicans ECE1 gene is highly inducedduring the conversion from noninvasive yeast growth to invasive hyphalgrowth. In order to clone Candida regulators of invasive growth, afusion between the ECE1 promoter and the E. coli lacZ gene wasconstructed. Specifically, PCR primers were designed to generate a DNAfragment consisting of 706 bases upstream of the ECE1 start codon andthe first 10 codons of the ECE1 gene. That fragment was cloned to createan in-frame translational fusion with lacZ carried on a yeast 2 μ/LEU2shuttle vector. The fusion plasmid was used to transform a diploidSaccharomyces cerevisiae strain that was homozygous leu2/leu2 andura3/ura3. The strain was subsequently transformed with a library ofCandida albicans genomic DNA fragments cloned in a yeast 2 μ/URA3vector. Resulting double transformants were recovered on selectivemedium lacking supplemental amino acids, pooled, and then replated atlow density (200-300 colonies/plate) on minimal medium that contained 50mM phosphate buffer (pH 7) and 0.003% w/v XGAL. The plates wereincubated for one week and dark blue colonies were picked with the ideathat cloned Candida regulators of invasion would activate transcriptionof lacZ via the ECE1 promoter. The 2 μ/URA3 plasmids were isolated fromthe blue transformants, retested and then the cloned DNA was sequenced.One of the genes identified was Candida AFL1. Expression of the CandidaAFL1 gene in S. cerevisiae imparted enhanced invasive behavior ondiploids under rich medium conditions where it normally does not invadethe substrate. Standard database searching for homologs of the CandidaAFL1 gene revealed an S. cerevisiae homolog. An S. cerevisiae straincontaining a complete deletion of AFL1 did not form filaments or invadeunder conditions where wild-type yeast did.

AFL1 regulates the expression of both S. cerevisiae FLO11 (describedabove) and Candida ECE1. It is likely that homologs of other S.cerevisiae genes which regulate FLO11 will regulate the promoters ofgenes which themselves regulate hyphal growth or invasion in otherfungi. The downstream genes can be determined through a number ofteclniques known to those skilled in the art. One example is to usemicroarray analysis, as described in DeRisi et al. Science 1997,278:680.

The use of Saccharomyces to clone a Candida gene by screening for itsfunction as an inducer of Candida ECE11 gene transcription exemplifiesanother approach to isolate important fungal genes involved in invasionor production of secondary metabolites. The success of this experimentin cloning an important Candida regulator of invasion proves the conceptthat it is possible to clone regulators of transcription frompotentially diverse species by using Saccharomyces as a heterologoushost in which to screen. This technique gives the advantages of thesophisticated molecular genetic tools available for Saccharomyces, butnot for other organisms. This has implications not only for fungi likeCandida, but potentially for very disparate species such as mammalianspecies. This method may also be useful in performing more detailedanalysis of the function of regulators of transcription fromnon-Saccharomyces species. The use of Saccharomyces as a heterologoushost can be combined with selection-based screens to rapidly screen cDNAor genomic libraries to isolate regulators of transcription of Candida,or any other organism.

Screening Assays for Genes and Compounds

As discussed above, we have identified a number of fungalinvasion-promoting and invasion-inhibiting factors that are involved inpathogenicity and that may therefore be used to screen for compoundsthat reduce the virulence of pathogenic fungi, as well as othermicrobial pathogens or, alternatively, increase the production ofsecondary metabolites. Any number of methods are available for carryingout such screening assays. According to one approach, candidatecompounds are added at varying concentrations to the culture medium ofpathogenic fungal cells expressing one of the nucleic acid sequencesdescribed herein. Gene expression is then measured, for example, bystandard northern blot analysis (Ausubel et al., sitpra), using anyappropriate fragment prepared from the nucleic acid molecule as ahybridization probe. The level of gene expression in the presence of thecandidate compound is compared to the level measured in a controlculture medium lacking the candidate molecule. A compound which promotesa decrease in the expression of the invasion-promoting factor isconsidered useful in the invention; such a molecule may be used, forexample, as a therapeutic to combat the pathogenicity of an infectiousorganism. A compound which promotes an increase in the expression of theinvasion-promoting factor is considered useful in the invention; such amolecule may be used, for example, as a potentiator, increasingproduction of commercially important secondary metabolites.

If desired, the effect of candidate compounds or genes may, in thealternative, be measured by assaying the protein polypeptide level.There are numerous preferred protein assays, including standardimmunological techniques, such as western blotting orimmunoprecipitation with an antibody specific for an invasinpolypeptide. For example, immunoassays may be used to detect or monitorthe expression of at least one of the polypeptides of the invention in apathogenic organism. Polyclonal or monoclonal antibodies (produced asdescribed in Harlow and Lane, Antibodies, a laboratory manual 1988, ColdSpring Harbor Press) which are capable of binding to such a polypeptidemay be used in any standard immunoassay format (e.g., ELISA, westernblot, or RIA assay) to measure the level of the pathogenicitypolypeptide. In another example, polypeptide levels may be determinedusing enzymatic activity in standard assays. Standard kits are available(for example the Galacto-Plus and GUS light systems, available fromTropix, Inc., Bedford Mass.) which allow for measurement of enzymaticactivity of, for example, Chloramphenicol acetyl transferase (CAT) andβ-galactosidase (encoded by the lacZ gene). Protein levels can also bedetermined using techniques such as fluorescence-activated cell sorting(FACS), spectrophotometry, or luminescence. A compound which promotes adecrease in the expression of the invasion-promoting polypeptide isconsidered useful. Again, such a molecule may be used, for example, as atherapeutic to combat the pathogenicity of an infectious organism. Acompound or which promotes an increase in the expression of theinvasion-promoting polypeptide is also considered useful. Again, such amolecule may be used, for example, as a potentiator, increasingproduction of commercially important secondary metabolites.

Alternatively, or in addition, candidate compounds may be screened forthose which specifically bind to and inhibit an invasin polypeptide ofthe invention. The efficacy of such a candidate compound is dependentupon its ability to interact with the invasin polypeptide. Such aninteraction can be readily assayed using any number of standard bindingtechniques and functional assays (e.g., those described in Ausubel etal., supra). For example, a candidate compound may be tested in vitrofor interaction and binding with a polypeptide of the invention and itsability to modulate pathogenicity may be assayed by any standard assays(e.g., those described herein).

In one particular example, a candidate compound that binds to a invasinpolypeptide may be identified using a chromatography-based technique.For example, a recombinant polypeptide of the invention may be purifiedby standard techniques from cells engineered to express the polypeptide(e.g., those described above) and may be immobilized on a column. Asolution of candidate compounds is then passed through the column, and acompound specific for the invasin polypeptide is identified on the basisof its ability to bind to the invasin polypeptide and be immobilized onthe column. To isolate the compound, the column is washed to removenon-specifically bound molecules, and the compound of interest is thenreleased from the column and collected. Compounds isolated by thismethod (or any other appropriate method) may, if desired, be furtherpurified (e.g., by high performance liquid chromatography). In addition,these candidate compounds may be tested for their ability to render apathogen less virulent (e.g., as described herein). Compounds isolatedby this approach may also be used, for example, as therapeutics to treator prevent the onset of a pathogenic infection, disease, or both.Compounds which are identified as binding to pathogenicity polypeptideswith an affinity constant less than or equal to 10 mM are consideredparticularly useful in the invention.

In yet another approach, candidate genes and compounds are screened forthe ability to inhibit the virulence of a fungal cell by monitoringexpression from a promoter element Inown to be positively or negativelyregulated during filamentous invasion, hyphal growth, pseudohyphalgrowth or haploid digging. These same selection-based systems may beconfigured to screen for genes or compounds which lead to increasedexpression from the FLO11 promoter (P_(FLO11)). In one example,P_(FLO11) is fused to a gene that confers a growth advantage when theirexpression is increased. For example, a P_(FLO11)-HIS3 fusion thatallows growth on SC-HIS when present in a his3 mutant has beengenerated. This fusion has the added advantage that expression levelscan be titrated by the compound 3-aminotriazole (3-AT). 3-AT is acompetitive inhibitor of His3 that, when present in sufficient amounts,will inhibit the His3 expressed from P_(FLO11) and prevent this strainfrom growing on SC-HIS. Therefore, growth of a strain containingP_(FLO11)-HIS3 only occurs on SC-HIS+3-AT plated when P_(FLO11)-HIS3expression is increased to overcome the competitive inhibition of His3by 3-AT. Increased expression of the transcription factor Tec1 confersincreased resistance to 3-AT in such a strain. By varying the amount of3-AT used, the extent of increased P_(FLO11)-HIS3 necessary for growthcan be modulated. Numerous markers could be used for such positiveselection systems, including, but not limited to, URA3, TRP1, ADE2,LEU2. Those skilled in the art will recognize that other positiveselection systems will also work in this system.

In another example, gene fusions that link P_(FLO11) to a gene productthat confers a growth disadvantage (in its most extreme case-death) canbe used to select for genes and compounds which down-regulate FLO11expression. In this case, any condition that decreases and/or eliminatesFLO11 expression will alleviate the growth disadvantage and allowstrains to grow. For example, a fusion of P_(FLO11) to the URA3 openreading frame that allows for growth on SC-URA in a P_(FLO11)-dependentmanner has been created. Since expression of URA3 is toxic in thepresence of 5-fluoro-orotic acid (5-FOA), selection against FLO11expression can be accomplished by screening for growth on SC+5-FOA of astrain carrying a P_(FLO11)URA3 fusion. Those skilled in the art willrecognize that other negative selection systems will also work in thissystem.

While the systems described above allow for the selection for or againstFLO11 expression, similar technologies can be used to screen for changesin FLO11 expression. Other examples of useful P_(FLO11) reporter genefusions that can be quantified include, but are not limited to,enzymatic or fluorescent reporters such as lacZ and green fluorescentprotein (GFP).

The selection and screening systems described here have a number ofpotential applications beyond those stated above. For example, they canbe used to identify, by selection methods, functional homologs for manyof the genes that regulate FLO11. For example, mutation of a gene thateliminates FLO11 expression in a strain deleted for his3 and carrying aP_(FLO11)HIS3 fusion will cause this strain to be a histidine auxotroph(unable to grow in media lacking histidine). However, cDNA or genomicDNA clones from other organisms that restore FLO11 expression in suchstrains (either by complementation or suppression of the mutant allele)can be directly selected for by selecting for growth on media lackinghistidine. Similarly, novel regulators of FLO11 expression from S.cerevisiae and heterologous organisms can be identified using the sameselection schemes.

In another application of using promoters known to be regulated duringhyphal or pseudohyphal growth, deletion constructs can be generated inorder to map the cis elements of the promoter required for each signaltransduction pathway. As an example, the FLO11 promoter screening andselection assays, in which nested deletions of 200 bp steps from the 3kb region upstream of the FLO11 ORF have been generated, are beinganalyzed for effects on lacZ expression in various wild-type and mutantbackground strains. In another example, overlapping 400 bp fragments ofthe FLO11 promoter have been cloned into the CYC1 transcriptional unit(FIG. 11). Together, these examples will demonstrate which DNA regionsare required for expression (and to what extent each of these regions isrequired). Furthermore, reporter gene fusions with subcloned pieces ofpromoters regulated during hyphal growth or pseudohyphal growth, such asthe FLO11 promoter, can be assessed for expression in various mutantsthat block (or activate) distinct pathways. A person skilled in the artwill recognize that regulatory elements from other invasins, includingthose described herein, can be isolated as described using the methodsdescribed herein, in conjunction with standard techniques described inAusubel et al. (supra). These methods include fusion of a DNA segmentfrom a putative regulatory or promoter region to a reporter gene, aswell as deletion analysis. Moreover, the pathway specificity of anyinvasin promoter can be determined through the use of wild-type andmutant host strains. If a given DNA segment confers reporter expressionin a wild-type strain, but shows either no or increased expression inspecific mutants, then this sequence can be inferred to mediate invasinexpression by whatever pathway that mutant defines. Using this approach,a set of gene fusions that report distinct pathways that regulate FLO11can be generated. A variety of basal or inducible promoter elements areapplicable for this technique, including, but not limited to, CYC1,PGK1, ADH1, GAL1-10, tet-R, MET25, and CUP1. Using the above system, amode of action can be assigned for any given treatment on FLO11expression. Assessment of the effect of a chemical or genetic treatmenton pathway-specific reporters will make it possible to determine whichpathway is altered by this treatment. For example, consider twodifferent reporter fusions, one (Reporter 1) that reports STEpathway-mediated expression of FLO11, and the other (Reporter 2) thatreports INV pathway-mediated expression of FLO11. If compound X, whichwas identified by its ability to eliminate expression of the full-lengthP_(FLO11), is shown to affect Reporter 1 but not Reporter 2, then it isinferred that compound X blocks FLO11 expression by affecting STEpathway activity. Similarly, where the compound affects a given pathwaywill also be refined using pathway-specific P_(FLO11) reporterconstructs and activated alleles of certain signaling components. Forexample, the mutant STE11-4 and overexpression of STE12 are bothpredicted to increase FLO11 expression. If compound X blocks theincreased expression of FLO11 mediated by STE11-4, but not STE12overexpression, then compound X can be inferred to antagonize the STEpathway before STE12, but at or below STE11. Assessing reporterexpression in various deletion mutants will enable similar approaches toassess the effect of compounds that activate expression of FLO11.

The reporters used in these experiments can be utilized in the same wayas those described for the full-length FLO11 promoter to allow forpositive and negative selections and a variety of screening methods.This will make it possible to identify pathway-specific modulators(heterologous genes, compounds, peptides etc.) of FLO11 expression. Sucha system is also useful in identifying modulators of important homologsof these regulatory genes from other organisms. For example, the smallG-protein Ras is activated in many kinds of human tumors, and it is ofinterest to identify inhibitors of RAS as candidate antitumor agents.Having a FLO11 promoter fragment fusion that reports the activity ofhuman RAS in yeast (e.g., in a yeast strain deleted for the two yeastRAS genes and expressing human RAS) would allow one to select and/orscreen for modulators of human Ras in yeast.

Another example of a screen is based on the observation that yeast fromhaploid S. cerevisiae invade agar when grown on rich (YPD) mediumwithout forming pseudohyphae. This nonfilamentous invasion behavior hasbeen termed “digging,” and the assay used to detect this behavior hasbeen termed the “dig assay.” Roberts and Fink, supra. Mutations thatblock digging of haploid S. cerevisiae on rich media also inhibitpseudohyphal growth and agar invasion of diploid S. cerevisiciae underlow nitrogen conditions. Mutant strains defective for digging arereferred to as “dig minus,” or “dig−.” The correlation of pseudohyphalinvasion and behavior in the dig assay is an essential aspect of thisscreen. The dig assay is comparatively easy to perform relative topseudohyphal invasion assays, and, as it occurs in haploid cells, ismore amenable to genetic approaches that rely upon recessive mutations.

We have discovered that pathogenic fungi can display similar diggingbehavior, characterized by increased invasion into or adherence to adefined substrate. For example, C. albicans displays a nonfilamentousinvasion of substrate in a variety of suitable conditions (e.g., YPD,and SPIDER). This behavior is similar to the reported digging behaviorof S. cerevisiae. As is true for S. cerevisiae, non-hyphal agar invasionby any given C. albicans mutant strain correlates with the ability ofthat strain to form filaments under filament-inducing conditions (e.g.,agar+serum). Thus, screening for compounds that block the diggingphenotype in fungi will allow for identification of compounds that blockfilament formation and, as a result, pathogenesis. These compounds areconsidered useful as fungicides or fungistats. Similarly, screening forcompounds that increase substrate invasion will allow for identificationof compounds which may also increase production or yield of secondarymetabolites.

An example of the dig assay is given below. To test whether a chemicalcompound would affect the nonfilamentous agar invasion behavior ofnonmutant S. cerevisiae or C. albicans at concentrations much lower thanthe lowest growth inhibitory concentration, these fungi were spottedonto agar plates containing rich medium (YPD) and a gradient of theantifungal fluconazole. The plates were then incubated for two days atoptimum growth temperatures and washed with water. Both fungi exhibitedinhibition of agar invasion at significantly lower concentrations offluconazole than those necessary to inhibit growth (FIG. 12). This assaycan be performed on other dimorphic fungi, such as U. maydis, as well ason other fungi. In all cases, the invasion assay can be used to isolatecompounds which inhibit invasion, or those which promote invasion. Eachtype of compound would have valued uses as described herein.

Optionally, invasion-inhibiting compounds identified in any of theabove-described assays may be confirmed as useful in conferringprotection against the development of a pathogenic infection in anystandard animal model. One example is described herein. Candidatecompounds, if successful, may be used for anti-pathogen therapeutics.Invasion-promoting compounds may be confirmed by assessing production ofsecondary metabolites. Candidate compounds, if successful, may be usedas potentiators to increase production or yield of these or othersecondary metabolites.

Test Compounds and Extracts

In general, compounds are identified from large libraries of bothnatural product and synthetic (or semi-synthetic) extracts or chemicallibraries according to methods known in the art. Those skilled in thefield of drug discovery and development will understand that the precisesource of test extracts or compounds is not critical to the screeningprocedure(s) of the invention. Accordingly, virtually any number ofchemical extracts or compounds can be screened using the methodsdescribed herein. Examples of such extracts or compounds include, butare not limited to, plant-, fungal-, prokaryotic- or animal-basedextracts, fermentation broths, and synthetic compounds, as well asmodification of existing compounds. Numerous methods are also availablefor generating random or directed synthesis (e.g., semi-synthesis ortotal synthesis) of any number of chemical compounds, including, but notlimited to, saccharide-, lipid-, peptide-, and nucleic acid-basedcompounds. Synthetic compound libraries are commercially available fromBrandon Associates (Merrimack, N.H.) and Aldrich Chemical (Milwaukee,Wis.). Alternatively, libraries of natural compounds in the form ofbacterial, fungal, plant, and animal extracts are commercially availablefrom a number of sources, including Biotics (Sussex, UK), Xenova(Slough, UK), Harbor Branch Oceangraphics Institute (Ft. Pierce, Fla.),and PharmaMar, U.S.A. (Cambridge, Mass.). In addition, natural andsynthetically produced libraries are produced, if desired, according tomethods known in the art, e.g., by standard extraction and fractionationmethods. Furthermore, if desired, any library or compound is readilymodified using standard chemical, physical, or biochemical methods.

In addition, those skilled in the art of drug discovery and developmentreadily understand that methods for dereplication (e.g., taxonomicdereplication, biological dereplication, and chemical dereplication, orany combination thereof) or the elimination of replicates or repeats ofmaterials already known for their anti-pathogenic activity should beemployed whenever possible.

When a crude extract is found to have an invasion-promoting orinvasion-inhibiting activity, or a binding activity, furtherfractionation of the positive lead extract is necessary to isolatechemical constituents responsible for the observed effect. Thus, thegoal of the extraction, fractionation, and purification process is thecareful characterization and identification of a chemical entity withinthe crude extract having the desired activity. Methods of fractionationand purification of such heterogenous extracts are known in the art. Ifdesired, compounds shown to be useful agents for the treatment ofpathogenicity or increased production of secondary metabolites arechemically modified according to methods known in the art.

Pharmaceutical Therapeutics and Plant Protectants The invention providesa simple means for identifying compounds (including peptides, smallmolecule inhibitors, and mimetics) capable of inhibiting thepathogenicity or virulence of a pathogen. Accordingly, chemical entitiesdiscovered to have medicinal or agricultural value using the methodsdescribed herein are useful as either drugs, plant protectants, or asinformation for structural modification of existing anti-pathogeniccompounds, e.g., by rational drug design. Such methods are useful forscreening compounds having an effect on a variety of pathogensincluding, but not limited to, bacteria, viruses, fungi, annelids,nematodes, Platyhelminthes, and protozoans. Examples of pathogenic fungiinclude, without limitation, Candida albicans, Aspergillus sp, Mucor sp,Rhizopus sp., Fusarium sp, Penicillium marneffei, Microsporum sp.Cryptococcis neoformans, Pneumocystis carinii, and Trichophyton sp.

For therapeutic uses, the compositions or agents identified using themethods disclosed herein may be administered systemically, for example,formulated in a pharmaceutically-acceptable buffer such as physiologicalsaline. Treatment may be accomplished directly, e.g., by treating theanimal with antagonists which disrupt, suppress, attenuate, orneutralize the biological events associated with a pathogenicitypolypeptide. Preferable routes of administration include, for example,inhalation or subcutaneous, intravenous, interperitoneally,intramuscular, or intradermal injections which provide continuous,sustained levels of the drug in the patient. Treatment of human patientsor other animals will be carried out using a therapeutically effectiveamount of an anti-pathogenic agent in a physiologically-acceptablecarrier. Suitable carriers and their formulation are described, forexample, in Remington's Pharmaceutical Sciences by E. W. Martin. Theamount of the anti-pathogenic agent to be administered varies dependingupon the manner of administration, the age and body weight of thepatient, and with the type of disease and extensiveness of the disease.Generally, amounts will be in the range of those used for other agentsused in the treatment of other microbial diseases, although in certaininstances lower amounts will be needed because of the increasedspecificity of the compound. A compound is administered at a dosage thatinhibits microbial proliferation. For example, for systemicadministration a compound is administered typically in the range of 0.1ng-10 g/kg body weight.

For agricultural uses, the compositions or agents identified using themethods disclosed herein may be used as chemicals applied as sprays ordusts on the foliage of plants, or in irrigation systems. Typically,such agents are to be administered on the surface of the plant inadvance of the pathogen in order to prevent infection. Seeds, bulbs,roots, tubers, and corms are also treated to prevent pathogenic attackafter planting by controlling pathogens carried on them or existing inthe soil at the planting site. Soil to be planted with vegetables,ornamentals, shrubs, or trees can also be treated with chemicalfumigants for control of a variety of microbial pathogens. Treatment ispreferably done several days or weeks before planting. The chemicals canbe applied by either a mechanized route, e.g., a tractor or with handapplications. In addition, chemicals identified using the methods of theassay can be used as disinfectants.

In addition, the antipathogenic agent may be added to materials used tomake catheters, including but not limited to intravenous, urinary,intraperitoneal, ventricular, spinal and surgical drainage catheters, inorder to prevent colonization and systemic seeding by potentialpathogens. Similarly, the antipathogenic agent may be added to thematerials that constitute various surgical prostheses and to dentures toprevent colonization by pathogens and thereby prevent more seriousinvasive infection or systemic seeding by pathogens.

Enhancement of Secondary Metabolite Production

The invention provides a simple means for identifying compounds(including peptides, small molecules and mimetics) that are capable ofactivating secondary metabolite production in fungi. Accordingly, achemical entity discovered to have such characteristics using themethods described herein is useful as a means to increase the yields ofcurrently marketed pharmaceuticals whose production, in whole or inpart, is dependent upon a fungal fermentation. Examples of marketedsecondary metabolites whose yields during fermentation could beincreased by such compounds include, without limitation, cyclosporin,penicillin, cephalosporin, ergot alkaloids, lovastatin, mevastatin, andthe biosynthetic intermediates thereof. In addition, such chemicalentities can also be used to increase the likelihood of identifying newsecondary metabolites with medicinal or agricultural value by increasingthe concentration of such metabolites (and hence, the likelihood ofdetection by chemical or bioassay) in a fermentation broth. In bothinstances, increased yield of metabolite can be accomplished bycontacting fungi with the entity and/or transforming a fungus with agene expression construct or constructs that would result in theproduction of said entity within the fungus.

The invention also provides a simple means for identifying candidategenes that both positively and negatively regulate secondary metaboliteproduction in fungi. In addition, methods to identify activated anddominant negative versions of these genes are described. Accordingly,such genes and their derivatives are likely candidates to enable thegenetic engineering of fungi to increase production of secondarymetabolites both through the introduction of altered forms of thesegenes into production fungi by standard transformation methods and alsoby elimination of relevant gene function through the construction ofknockouts. Examples of marketed secondary metabolites whose yieldsduring fermentation could be increased by genetic engineering methodssuch as these include, without limitation, cyclosporin, penicillin,cephalosporin, ergot alkaloids, lovastatin, mevastatin, and thebiosynthetic intermediates thereof.

Production and Detection Methods for Fungal Secondary Metabolites

Methods for fermentation and production of beta-lactam antibiotics,statins, ergot alkaloids, cyclosporin, and other fungal metabolites aredescribed in Masurekar (Biotechnology 1992, 21: 241-301), and referencestherein. The detection of secondary metabolites is specific for eachmetabolite and well-known to those practiced in the art. General methodsto assess production and integrity of compounds in fermentation brothsinclude, but are not limited to, bioassays for antimicrobial activity,high-performance liquid chromatography (HPLC) analysis, nuclear magneticresonance, thin-layer chromatography, and absorbance spectrometry.Purification of metabolites from a fermentation broth can includeremoval of fungal cells or hyphae by centrifugation or filtration,adjustment of pH and/or salt concentrations after fermentation (toenhance solubility and/or subsequent extraction efficiency), andextraction of broths with appropriate organic solvents.

Other Embodiments

In general, the invention includes any nucleic acid sequence which maybe isolated as described herein or which is readily isolated by homologyscreening or PCR amplification using the nucleic acid sequences of theinvention. Also included in the invention are polypeptides which aremodified in ways which do not abolish their pathogenic activity(assayed, for example as described herein). Such changes may includecertain mutations, deletions, insertions, or post-translationalmodifications, or may involve the inclusion of any of the polypeptidesof the invention as one component of a larger fusion protein.

Thus, in other embodiments, the invention includes any protein which issubstantially identical to a polypeptide of the invention. Such homologsinclude other substantially pure naturally-occurring polypeptides aswell as allelic variants; natural mutants; induced mutants; proteinsencoded by DNA that hybridizes to any one of the nucleic acid sequencesof the invention under high stringency conditions or, less preferably,under low stringency conditions (e.g., washing at 2×SSC at 40° C. with aprobe length of at least 40 nucleotides); and proteins specificallybound by antisera of the invention.

The invention further includes analogs of any naturally-occurringpolypeptide of the invention. Analogs can differ from thenaturally-occurring polypeptide of the invention by amino acid sequencedifferences, by post-translational modifications, or by both. Analogs ofthe invention will generally exhibit at least 85%, more preferably 90%,and most preferably 95% or even 99% identity with all or part of anaturally-occurring amino acid sequence of the invention. The length ofsequence comparison is at least 15 amino acid residues, preferably atleast 25 amino acid residues, and more preferably more than 35 aminoacid residues. Again, in an exemplary approach to determining the degreeof identity, a BLAST program may be used, with a probability scorebetween e⁻³ and e⁻¹⁰⁰ indicating a closely related sequence.Modifications include in vivo and in vitro chemical derivatization ofpolypeptides, e.g., acetylation, carboxylation, phosphorylation, orglycosylation; such modifications may occur during polypeptide synthesisor processing or following treatment with isolated modifying enzymes.Analogs can also differ from the naturally-occurring polypeptides of theinvention by alterations in primary sequence. These include geneticvariants, both natural and induced (for example, resulting from randommutagenesis by irradiation or exposure to ethanemethylsulfate or bysite-specific mutagenesis as described in Sanbrook, Fritsch andManiatis, Molecular Cloning: A Laboratory Manual (2d ed.), CSH Press,1989, or Ausubel et al., supra). Also included are cyclized peptides,molecules, and analogs which contain residues other than L-amino acids,e.g., D-amino acids or non-naturally occurring or synthetic amino acids,e.g., β or γ amino acids.

In addition to full-length polypeptides, the invention also includesfragments of any one of the polypeptides of the invention. As usedherein, the term “fragment,” means at least five, preferably at least 20contiguous amino acids, preferably at least 30 contiguous amino acids,more preferably at least 50 contiguous amino acids, and most preferablyat least 60 to 80 or more contiguous amino acids. Fragments of theinvention can be generated by methods known to those skilled in the artor may result from normal protein processing (e.g., removal of aminoacids from the nascent polypeptide that are not required for biologicalactivity or removal of amino acids by alternative mRNA splicing oralternative protein processing events).

The invention further provides compositions (e.g., nucleotide sequenceprobes) and methods for the diagnosis of a pathogenic condition.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindependent publication or patent application was specifically andindividually indicated to be incorporated by reference.

Other embodiments are within the scope of the claims.

67 1 39 DNA Artificial Sequence oligonucleotide primer 1 gaattaaccctcactaaagg gaaarmgnga ycayathac 39 2 38 DNA Artificial Sequenceoligonucleotide primer 2 gtaatacgac tcactatagg gtgyttyttn arrtcytg 38 33463 DNA Candida RIM1 3 ttaaaaagtt tttgattgtt gaacttttaa attttttcttggcaatccat tcccagacaa 60 agtaataact acgaatagat cattcattgg tttattatttttgcatggaa atatttgaat 120 ttccattttt tttttttata gtggttgttt aagcttcgcagttttttttt ttctagggag 180 aaattattat acattatata tatattatca actttttctcgttacaaaag tcacaccttt 240 tttttttcta cttgttcttc tttcaacaac taactaattttatactatcc acgaactata 300 gatattacat ataagttttt aacctagaca aacgagatttttagacaatg aattacaaca 360 ttcatcccgt aacatactta aatgctgata gcaataccggtgcaagtgag agtactgcaa 420 gtcaccatgg ttccaagaaa tcaccttcct cagatattgatgtagataat gctwcgtcac 480 cttcatcttt tacttcgtcc caatcacctc acattaatgctatgggtaac agtccccatt 540 cctcattcac ttctcaatct gcagccaatt ctcctatcactgatgccaaa caacatttgg 600 ttaaaccaac caccaccaag ccagcagctt ttgctcctagtgctaatcaa tctaacacca 660 cagctccgca atcttatacc caaccagcac aacaattaccaactcagtta cacccaagtc 720 ttaaccaagc ctacaacaac caaccatctt attatttacaccaaccaact tatggctacc 780 aacaacaaca acaacaacaa caacaccaag agtttaaccaaccatcacag caataccacg 840 accatcacgg atactactca aacaacaaca ttttgaatcagaatcaacca gctccacaac 900 aaaatccagt caagccattc aaaaagacat acaagaaaatcagagacgaa gatttgaaag 960 gtcctttcaa gtgtttgtgg agcaactgta gcattattttcgagactcca gaaattttgt 1020 acgatcattt gtgtgacgac catgttggta gaaagtcttcgaacaatttg tcattgactt 1080 gtctttggga aaattgtggc acaactacag ttaagagagatcacattact tctcacttga 1140 gagtccatgt cccattgaag cctttccatt gtgacttgtgtcccaaatcg ttcaagagac 1200 ctcaagattt gaagaaacat tccaagactc acgctgaagaccatccaaag aagttaaaaa 1260 aggcacaaag agagttgatg aaacaacaac aaaaagaggccaagcaacaa cagaaattgg 1320 ccaacaagag agcaaactcg atgaatgcaa ctaccgcatccgatttgcaa ttgaactact 1380 attctggtaa ccctgctgat ggattgaact acgacgacacctccaaaaaa agaagatacg 1440 aaaacaattc tcaacacaac atgtatgtgg ttaatagtattttgaacgat ttcaacttcc 1500 aacaaatggc tcaagctcca cagcaaccag gcgttgttggaaccgcaggt tctgctgagt 1560 tcaccaccaa gaggatgaaa gccggcactg agtataacattgatgtgttt aacaagttga 1620 atcatttgga cgaccacttg caccaccacc accctcaacagcaacaccca caacaacaat 1680 atggcggtaa catctatgaa gctgaaaaat tcttcaactccttatcgaat tccatcgaca 1740 tgcaatatca aaacatgtca acccaatatc aacaacaacatgctggttct acttttgctc 1800 aacagaaacc aactcaacaa gcaagtggcc aattgtatccttctttacca accattggca 1860 atggctcata caccagtgga tcatcacaca aagaagggttggttaataac cataacggat 1920 acttgccatc ttatcctcaa atcaaccgtt ctttgccatattcttctggt gtggcacaac 1980 aaccaccaag tgcattagag tttggcggtg tttcaacctaccagaaatct gcacaatcat 2040 atgaagaaga cagcagcgac agttcagagg aagacgattacagcacttct tcagaagacg 2100 agcttgacac cttgtttgat aaattaaaca tcgatgacaataaagttgaa gaagtgacga 2160 ttgatgggtt caatttgaag gatgttgcca agcacagagaaatgatccat gctgttcttg 2220 gctatttgag aaaccaaatc gaacaacaag aaaaggaaaagagcaaagaa caaaaggagg 2280 ttgacgttaa tgaaactaaa ttatatccaa ctataactgctttctaagca attatatcga 2340 ttttactttt ttatttattt ttattttttt gtttagggtggttttcaatt tttttttttt 2400 atttcctcat gtttgatttt agtgtgtttt attgtatattacgtataagt ttattttatt 2460 agtacaagtt ttgaaagtag tgttaccgtt ctctatttacatggttctat taatcattcc 2520 acctcccaat acttgattcc ctttgtacaa cacaccagcttgacctggcg ccaaagcacg 2580 aactggggtt tctaattcga cggttaacct atcttgcagctcaataattc ttgtaacagg 2640 gattgacttc agcaatgaat gatactgaaa ttgcaatgattggagacaaa acaccttttc 2700 cttagggtgt aaccattcta actgcgtcaa ttccacagtttgtttgaaca acttggggtt 2760 atcgtgacct ttaactatga tgatttgatt tttctcataattcttatcgc tgacaaacca 2820 aatgccttgg tattgtggat ctgcttgcgg catgcacactgaagatttct ggcctatcgt 2880 agcatgccat agccccttgt gttgacccca aaccttaccatcttcagtga ttatgtcacc 2940 tgggttttct gggatataat catttaaaaa ctctcggaagtttgactgct gcggattaac 3000 aaagcatagc ccttgggagt cgggttttgt tgctgtgtgtaatttaaact ggtcatgtgc 3060 caattcccga atctgtggtt tgatatagtg gccaatgggtaataagattt tcgataacga 3120 agattgtggg atcgacgaaa gatagtaact ttggtctttccgttgactca aacctcgcaa 3180 taaatggtac tctccagttt cgttgtgttt catgattcgggcgtaatgac cagtgactaa 3240 ccaccaatct ttgcctgtgc catcgtattt cttatgcaagtaatcaatca acttcccaaa 3300 tttgacaaat ttgttgcacc caatgtcagg gttcggcgttagtccctttt cgtatttctc 3360 tatcattggc ataaacacat cctgccaata ttcgtgttcaaagttaactc tctcacaact 3420 ggaaatacct agatcgacct cgaggggggg cccggtaccagtt 3463 4 659 PRT Candida RIM1 MISC_FEATURE (43)..(43) Amino acid 43 is“Xaa” wherein “Xaa” = any amino acid. 4 Met Asn Tyr Asn Ile His Pro ValThr Tyr Leu Asn Ala Asp Ser Asn 1 5 10 15 Thr Gly Ala Ser Glu Ser ThrAla Ser His His Gly Ser Lys Lys Ser 20 25 30 Pro Ser Ser Asp Ile Asp ValAsp Asn Ala Xaa Ser Pro Ser Ser Phe 35 40 45 Thr Ser Ser Gln Ser Pro HisIle Asn Ala Met Gly Asn Ser Pro His 50 55 60 Ser Ser Phe Thr Ser Gln SerAla Ala Asn Ser Pro Ile Thr Asp Ala 65 70 75 80 Lys Gln His Leu Val LysPro Thr Thr Thr Lys Pro Ala Ala Phe Ala 85 90 95 Pro Ser Ala Asn Gln SerAsn Thr Thr Ala Pro Gln Ser Tyr Thr Gln 100 105 110 Pro Ala Gln Gln LeuPro Thr Gln Leu His Pro Ser Leu Asn Gln Ala 115 120 125 Tyr Asn Asn GlnPro Ser Tyr Tyr Leu His Gln Pro Thr Tyr Gly Tyr 130 135 140 Gln Gln GlnGln Gln Gln Gln Gln His Gln Glu Phe Asn Gln Pro Ser 145 150 155 160 GlnGln Tyr His Asp His His Gly Tyr Tyr Ser Asn Asn Asn Ile Leu 165 170 175Asn Gln Asn Gln Pro Ala Pro Gln Gln Asn Pro Val Lys Pro Phe Lys 180 185190 Lys Thr Tyr Lys Lys Ile Arg Asp Glu Asp Leu Lys Gly Pro Phe Lys 195200 205 Cys Leu Trp Ser Asn Cys Ser Ile Ile Phe Glu Thr Pro Glu Ile Leu210 215 220 Tyr Asp His Leu Cys Asp Asp His Val Gly Arg Lys Ser Ser AsnAsn 225 230 235 240 Leu Ser Leu Thr Cys Leu Trp Glu Asn Cys Gly Thr ThrThr Val Lys 245 250 255 Arg Asp His Ile Thr Ser His Leu Arg Val His ValPro Leu Lys Pro 260 265 270 Phe His Cys Asp Leu Cys Pro Lys Ser Phe LysArg Pro Gln Asp Leu 275 280 285 Lys Lys His Ser Lys Thr His Ala Glu AspHis Pro Lys Lys Leu Lys 290 295 300 Lys Ala Gln Arg Glu Leu Met Lys GlnGln Gln Lys Glu Ala Lys Gln 305 310 315 320 Gln Gln Lys Leu Ala Asn LysArg Ala Asn Ser Met Asn Ala Thr Thr 325 330 335 Ala Ser Asp Leu Gln LeuAsn Tyr Tyr Ser Gly Asn Pro Ala Asp Gly 340 345 350 Leu Asn Tyr Asp AspThr Ser Lys Lys Arg Arg Tyr Glu Asn Asn Ser 355 360 365 Gln His Asn MetTyr Val Val Asn Ser Ile Leu Asn Asp Phe Asn Phe 370 375 380 Gln Gln MetAla Gln Ala Pro Gln Gln Pro Gly Val Val Gly Thr Ala 385 390 395 400 GlySer Ala Glu Phe Thr Thr Lys Arg Met Lys Ala Gly Thr Glu Tyr 405 410 415Asn Ile Asp Val Phe Asn Lys Leu Asn His Leu Asp Asp His Leu His 420 425430 His His His Pro Gln Gln Gln His Pro Gln Gln Gln Tyr Gly Gly Asn 435440 445 Ile Tyr Glu Ala Glu Lys Phe Phe Asn Ser Leu Ser Asn Ser Ile Asp450 455 460 Met Gln Tyr Gln Asn Met Ser Thr Gln Tyr Gln Gln Gln His AlaGly 465 470 475 480 Ser Thr Phe Ala Gln Gln Lys Pro Thr Gln Gln Ala SerGly Gln Leu 485 490 495 Tyr Pro Ser Leu Pro Thr Ile Gly Asn Gly Ser TyrThr Ser Gly Ser 500 505 510 Ser His Lys Glu Gly Leu Val Asn Asn His AsnGly Tyr Leu Pro Ser 515 520 525 Tyr Pro Gln Ile Asn Arg Ser Leu Pro TyrSer Ser Gly Val Ala Gln 530 535 540 Gln Pro Pro Ser Ala Leu Glu Phe GlyGly Val Ser Thr Tyr Gln Lys 545 550 555 560 Ser Ala Gln Ser Tyr Glu GluAsp Ser Ser Asp Ser Ser Glu Glu Asp 565 570 575 Asp Tyr Ser Thr Ser SerGlu Asp Glu Leu Asp Thr Leu Phe Asp Lys 580 585 590 Leu Asn Ile Asp AspAsn Lys Val Glu Glu Val Thr Ile Asp Gly Phe 595 600 605 Asn Leu Lys AspVal Ala Lys His Arg Glu Met Ile His Ala Val Leu 610 615 620 Gly Tyr LeuArg Asn Gln Ile Glu Gln Gln Glu Lys Glu Lys Ser Lys 625 630 635 640 GluGln Lys Glu Val Asp Val Asn Glu Thr Lys Leu Tyr Pro Thr Ile 645 650 655Thr Ala Phe 5 4792 DNA Saccharomyces cerevisiae INV9 5 cccagtttctcataacccgt catttgagcg attgctagtt gaaactatat acagctagtt 60 agtaagattttaaaagacga aaagaacgga aagtaggaca actaccaaaa aaaaagatgg 120 aaggtttctttggttagtta gcagtatccg cgtaatgcct tagacgtttt tgatcagaaa 180 aaaataaaacttgatttcgc ccaggatcga actggggacg ttctgcgtgt taagcagatg 240 ccataaccgactagaccacg aaaccagttt tttgatgttt cgtttaggac aagagtccta 300 tgagagtccactaaaattct aaaaataatt gtatcaacaa ccgtcatgca gctgttgtat 360 caagaatcgaccaccatcta tagactattg ttcatagctg tattacaata tcatataaga 420 tgtaaggatagaacgtgaag atcgagaaac agtcagcaga tttaatggaa gctgaaatgc 480 acggattgataatgtaatag aataagtgac aacataagaa atgaaagaaa aaaaataaca 540 ttaatataatttagaaatgt tagtttcctt ctatgaactc tcgtatccct ggggaggact 600 ttcaatatattcagtatacc taccgttcaa atgatactct aaaatatcat ctattaagtg 660 gtattcaggtttttcccacg caagattgag tgcgtcaggc aaagccacga tcgcagatta 720 ctctcctaacaatattaaga tcgtcaagaa catccggttt tactcaagcg cttaatacaa 780 cacgtcggctttcgctcaac tgcagtatat tgtgtctgta acctctcccg tcaagtttgc 840 gccacgggcccaataaccaa cttctgcgcc attgttacgg aaaagtggcc atctttgcta 900 tgctcgtaaggctagcgctc tctaagtacg ccagcccaaa aactccgtat cgctcttcat 960 cggaattgctatatctacag ccggacgggc actctatgta tactcatatc acagccactg 1020 ttgcactacattattcgcag ggactccgag atcgctgagc atgttgctaa gcaacttacc 1080 aaggttggaatgcattatat agtccgatat agaccccgct cggactatat tatataaatt 1140 caaatagcactttcaacgag tgtcttaatc cgtcctagtt cctgctctcg ctccacgttg 1200 tcatcccacccattcgcacg ggtcttctgt gcgaatgagc cacaacgggg ccgagttcag 1260 gccgtgtccgcctacaacgt ccgccaacta gtggcaattg ctacgtacgc cgaccacgct 1320 gacacgcaccgttctactcg ctctatgctg cggtgtgacg tgtgaacgcg cgatcgttcg 1380 caatcgtatcgtttgccaag aaaaaaagcc aatcggagaa tctgaaaatc gctattcggc 1440 ttgcaggtcgcacgcactca gagtacgaaa tgccaagtac cggaatttcc ttggcctttt 1500 ttaagtttcttctcttctat tctttttccc ctttctttct tcctttgcta tctgtctggt 1560 ttaaataaacatagtatttt tttgtgtcta agcctcttcc tcttctctcg tacttgctct 1620 actaccactttacttaatcg cctttctttg ttttctttct tcgttatttg ttcttggaac 1680 ttttccgctccaatcccaac gattggcttc aaaacacgtt ctactgtcta gcaatttctg 1740 caggtgccatttttcttagg cttataccct ttccttttcc ccttacattt gatttcttct 1800 tcaaagttccttatagtatt attgtctaag ctctattgag tcaaaagtaa caatctagac 1860 gaaggaaaaaaaaaaaatag aaaatagaca tccccgaata cgcatcatct cacgcacgta 1920 caagattttaacgttaaagc caaagtacgc tagtatagta tcatcagcat caccctcact 1980 atcggtagcattgaccaaac atgtcgttac tgagactgtg gaacaaagaa tcaagggcac 2040 catcaaaaataaagagtcat ggtattgttg gcagttacgg caacagcatg ctggcccata 2100 acaacgtgaagcaatttcgt atagacatag acgaaccgca tagagtatgg aaaccgaatg 2160 aaagcataaccggagaagcg gtcattgaca taaagagaga cataactaac gtagcgatca 2220 aattatcgctagtatgtgag gttcgcgtga aaacggggaa cagtccaacc tccaagaata 2280 agagaattgagaaaacctta gagaagtcga cgtttcttta tggacaggac tacgtaaaga 2340 cagctttttcggctaaggaa aagaaaccgc atgttgacaa aaccaccatt ctcaatggtt 2400 taagcaagggggaacacagg tttcccttta ggatacgaat accacgaggc agaggaatgt 2460 tgagctctataaagttcgaa aggggctcga taacatactt cctctcttgc actttagaat 2520 ccctcaacaacatcaacgga ttaaaaaaac cggaagcaag atgcgaacgt gagtttgcag 2580 tcatagttccgctggacgtc tcgaggctgc ccaagccgaa aactaagaca gtggttttac 2640 aatcagcatctatggtccaa aacaaaaaga acaaatctac agaggacgaa tcctcatcgt 2700 atacacaattaactcaaaag tctactactt ctaattcttc tagcagttca gtaaactcca 2760 agacgtcccccttaccaaat aaaacggtga ctatatccgt agacataccg caggctggat 2820 tcatgattggtgaaattatc cctatagacg ttaagattga ccactataag cctttctatg 2880 cccctgcgggtctcaccacc actttggtga ggatatgtag ggtgggcggt gcaggcaaag 2940 atgatcctatggagactttc agaaaagata tatgtcagag tatctctcct atatatatta 3000 accctgaaacgttgcagttt caatctagag tttatctgaa agtgcccctt gatgcatttt 3060 cgacccttactactgtggga aaatttttct ccttccaata ctatatcgag gttatggtta 3120 acttatcaaaaaaaaacgtg gtttacacag aatctaatag aataatagga actcctattg 3180 gagaacaaaatggcttgggc gtagagaata atatcaaccg tatccaaagg aaaatgctac 3240 gtatggtcaatccagaaacg ttggagaacg attctgaggg ttatgaatcc agtatatttt 3300 tcaaagatatggtaaatgtg gaaaagctaa agagactgag gaatgtaact ggtatgtcca 3360 tagaaaccgtcataggaacg acgagatccg aacagcagca atctgatgca agcatcccat 3420 cccaatcctcaatcacggct cctcaaaatt ctccatcgaa tttaagagat tggttggccc 3480 cattaaatgcatatgatagt gacgatgttc cagttccaaa gtattcgcca aatgataaag 3540 tcagtgtaccgtcggaagac aaacaagaac ttgaacaaaa aagactacaa cagttagaaa 3600 gcgatcctcccccttgtgat gactattaaa aagtgcaggt aacaagtcat atactcgcag 3660 cttgcgccgtgttggaacta ggcgccttaa tcatgtttgc atatttccac tatcccagcc 3720 acgtaatgatccatgacatt aacatagaaa aaaaaaatcg aagcatgcac aaacctgaga 3780 tttatatatgttcatgtgta cttaatatac gtttaatgat taaaactata gccgtcctca 3840 ggcaaactgagataagaaac gaaaaaatag cagtaacgta aacgttattc tatatttata 3900 aagacgtcaaaaaaaaaagt gattgtgata ttgagatgta agctatatac cgaactttga 3960 gctccctcacgtggaaaata tgatagattg ttgcctcatc attgcggaac cgcatttttt 4020 ttttgtatttttgcctccct agtttcaaaa tgcaccaaat tctcccctta atgctttttg 4080 ttttaagtcccaaatagcca tcctttcatc atcgggcaag atagaaattt gactgtcatt 4140 cagttgtaacacctgtttca gtagttcttt ctgtttgtta agcacagctg gatccaccac 4200 ctcgttcacgctattgttat tcgtagccga tgcctcttct tgcggcctgg aagctaacgg 4260 gatcaaatcatccactttac atatcccatt cgttagcaat aattcagccg taacaaaact 4320 caactgtggacacagctcta atagcgaaac agcatcttca ggatgcgctc ttgtccattc 4380 ttggaatttttgtaaaaatt tcaactgcac ctctttcggt tttttagcta gttcgctcga 4440 tatcatcatagcaggggtgg tcatgtttat gttaacgtcg ataccagagg gcaattctgg 4500 aaacttttgacttagaaaat tggcatttcc gctgttttga aagtccggcc cattactatt 4560 attattattatttccattgt tgttattgtt cccattaatg ttgttgtact gttgttgttg 4620 ctgttgtgaaactcccgata tatcactatt gctggagtaa ccgcatttca aaaacctaga 4680 gcctaattggtatccattca aattacgtac tgcgctggca ctggactcta aatctctaaa 4740 ttcaataaacgcgtaccctt tcgacctacc agtttggggg tcgaacatca tt 4792 6 542 PRTSaccharomyces cerevisiae INV9 6 Met Ser Leu Leu Arg Leu Trp Asn Lys GluSer Arg Ala Pro Ser Lys 1 5 10 15 Ile Lys Ser His Gly Ile Val Gly SerTyr Gly Asn Ser Met Leu Ala 20 25 30 His Asn Asn Val Lys Gln Phe Arg IleAsp Ile Asp Glu Pro His Arg 35 40 45 Val Trp Lys Pro Asn Glu Ser Ile ThrGly Glu Ala Val Ile Asp Ile 50 55 60 Lys Arg Asp Ile Thr Asn Val Ala IleLys Leu Ser Leu Val Cys Glu 65 70 75 80 Val Arg Val Lys Thr Gly Asn SerPro Thr Ser Lys Asn Lys Arg Ile 85 90 95 Glu Lys Thr Leu Glu Lys Ser ThrPhe Leu Tyr Gly Gln Asp Tyr Val 100 105 110 Lys Thr Ala Phe Ser Ala LysGlu Lys Lys Pro His Val Asp Lys Thr 115 120 125 Thr Ile Leu Asn Gly LeuSer Lys Gly Glu His Arg Phe Pro Phe Arg 130 135 140 Ile Arg Ile Pro ArgGly Arg Gly Met Leu Ser Ser Ile Lys Phe Glu 145 150 155 160 Arg Gly SerIle Thr Tyr Phe Leu Ser Cys Thr Leu Glu Ser Leu Asn 165 170 175 Asn IleAsn Gly Leu Lys Lys Pro Glu Ala Arg Cys Glu Arg Glu Phe 180 185 190 AlaVal Ile Val Pro Leu Asp Val Ser Arg Leu Pro Lys Pro Lys Thr 195 200 205Lys Thr Val Val Leu Gln Ser Ala Ser Met Val Gln Asn Lys Lys Asn 210 215220 Lys Ser Thr Glu Asp Glu Ser Ser Ser Tyr Thr Gln Leu Thr Gln Lys 225230 235 240 Ser Thr Thr Ser Asn Ser Ser Ser Ser Ser Val Asn Ser Lys ThrSer 245 250 255 Pro Leu Pro Asn Lys Thr Val Thr Ile Ser Val Asp Ile ProGln Ala 260 265 270 Gly Phe Met Ile Gly Glu Ile Ile Pro Ile Asp Val LysIle Asp His 275 280 285 Tyr Lys Pro Phe Tyr Ala Pro Ala Gly Leu Thr ThrThr Leu Val Arg 290 295 300 Ile Cys Arg Val Gly Gly Ala Gly Lys Asp AspPro Met Glu Thr Phe 305 310 315 320 Arg Lys Asp Ile Cys Gln Ser Ile SerPro Ile Tyr Ile Asn Pro Glu 325 330 335 Thr Leu Gln Phe Gln Ser Arg ValTyr Leu Lys Val Pro Leu Asp Ala 340 345 350 Phe Ser Thr Leu Thr Thr ValGly Lys Phe Phe Ser Phe Gln Tyr Tyr 355 360 365 Ile Glu Val Met Val AsnLeu Ser Lys Lys Asn Val Val Tyr Thr Glu 370 375 380 Ser Asn Arg Ile IleGly Thr Pro Ile Gly Glu Gln Asn Gly Leu Gly 385 390 395 400 Val Glu AsnAsn Ile Asn Arg Ile Gln Arg Lys Met Leu Arg Met Val 405 410 415 Asn ProGlu Thr Leu Glu Asn Asp Ser Glu Gly Tyr Glu Ser Ser Ile 420 425 430 PhePhe Lys Asp Met Val Asn Val Glu Lys Leu Lys Arg Leu Arg Asn 435 440 445Val Thr Gly Met Ser Ile Glu Thr Val Ile Gly Thr Thr Arg Ser Glu 450 455460 Gln Gln Gln Ser Asp Ala Ser Ile Pro Ser Gln Ser Ser Ile Thr Ala 465470 475 480 Pro Gln Asn Ser Pro Ser Asn Leu Arg Asp Trp Leu Ala Pro LeuAsn 485 490 495 Ala Tyr Asp Ser Asp Asp Val Pro Val Pro Lys Tyr Ser ProAsn Asp 500 505 510 Lys Val Ser Val Pro Ser Glu Asp Lys Gln Glu Leu GluGln Lys Arg 515 520 525 Leu Gln Gln Leu Glu Ser Asp Pro Pro Pro Cys AspAsp Tyr 530 535 540 7 29 DNA Artificial Sequence PCR primer 7 cgcacactatgcaaagaccg agatcttcc 29 8 29 DNA Artificial Sequence PCR primer 8gaagatcttc tccacatacc aatcactcg 29 9 40 DNA Artificial Sequence #1Fprimer 9 caagcattta cgttactgcg aaaatccata tacgcacact 40 10 40 DNAArtificial Sequence #1R primer 10 agtgtgcgta tatggatttt cgcagtaacgtaaatgcttg 40 11 40 DNA Artificial Sequence #2F primer 11 tgatgaggtaacctttacaa ctctcttcta gttcaagaac 40 12 40 DNA Artificial Sequence #2Rprimer 12 gttcttgaac tagaagagag ttgtaaaggt tacctcatca 40 13 40 DNAArtificial Sequence #3F primer 13 tttcaattca atggatttgg aatgtcatagagttaccaat 40 14 40 DNA Artificial Sequence #3R primer 14 attggtaactctatgacatt ccaaatccat tgaattgaaa 40 15 40 DNA Artificial Sequence #4Fprimer 15 atttctgcct atactcttaa aggtattcgt ttgtttacta 40 16 40 DNAArtificial Sequence #4R primer 16 tagtaaacaa acgaatacct ttaagagtataggcagaaat 40 17 40 DNA Artificial Sequence #5F primer 17 ttggggctaagaatggacac agatcagtca ttcatgttgt 40 18 40 DNA Artificial Sequence #5Rprimer 18 acaacatgaa tgactgatct gtgtccattc ttagccccaa 40 19 40 DNAArtificial Sequence #6F primer 19 gggtgtgcct ggaaagttca ttcccttttctttttctttg 40 20 40 DNA Artificial Sequence #6R primer 20 caaagaaaaagaaaagggaa tgaactttcc aggcacaccc 40 21 40 DNA Artificial Sequence #7Fprimer 21 caaaacttta ggaataccgg aaattaaggt ttttttcttc 40 22 40 DNAArtificial Sequence #7R primer 22 gaagaaaaaa accttaattt ccggtattcctaaagttttg 40 23 40 DNA Artificial Sequence #8F primer 23 cgaatgtgaatgcgctaatc ttgtgtgcct acgccagccc 40 24 40 DNA Artificial Sequence #8Rprimer 24 gggctggcgt aggcacacaa gattagcgca ttcacattcg 40 25 40 DNAArtificial Sequence #9F primer 25 agacaaaaaa taggaaaagt ggtatttccaccacatgaaa 40 26 40 DNA Artificial Sequence #9R primer 26 tttcatgtggtggaaatacc acttttccta ttttttgtct 40 27 40 DNA Artificial Sequence #10Fprimer 27 ttagtgcgga atactttcct ttaattagtg atggttctca 40 28 40 DNAArtificial Sequence #10R primer 28 tgagaaccat cactaattaa aggaaagtattccgcactaa 40 29 40 DNA Artificial Sequence #11F primer 29 cagtgctttcaacacctttt attctcatcg agagccgagc 40 30 40 DNA Artificial Sequence #11Rprimer 30 gctcggctct cgatgagaat aaaaggtgtt gaaagcactg 40 31 40 DNAArtificial Sequence #12F primer 31 gtagctgaaa agtccatcta catctgtgtgccatgtcaga 40 32 40 DNA Artificial Sequence #12R primer 32 tctgacatggcacacagatg tagatggact tttcagctac 40 33 40 DNA Artificial Sequence #13Fprimer 33 gagattatct tgggatctat tcgaattatg aatgatacta 40 34 40 DNAArtificial Sequence #13R primer 34 tagtatcatt cataattcga atagatcccaagataatctc 40 35 40 DNA Artificial Sequence #14F primer 35 gttttggctcaatgggaccg ttcacaaatt tacggctaat 40 36 40 DNA Artificial Sequence #14Rprimer 36 attagccgta aatttgtgaa cggtcccatt gagccaaaac 40 37 70 DNAArtificial Sequence URA3 primer sequence 37 accacaacat gacgagggataataactgat gaatagggtg ctttttatac tctgtgcggt 60 atttcacacc 70 38 70 DNAArtificial Sequence URA3 primer sequence 38 taaggagtcg taccgccaactaaatctgaa taacaatttg gctgctagaa gcagattgta 60 ctgagagtgc 70 39 29 DNAArtificial Sequence #1R primer 39 ccgctcgaga gtgtgcgtat atggatttt 29 4029 DNA Artificial Sequence #2F primer 40 ccgctcgagt gatgaggtaa cctttacaa29 41 29 DNA Artificial Sequence #2R primer 41 ccgctcgagg ttcttgaactagaagagag 29 42 29 DNA Artificial Sequence #3F primer 42 ccgctcgagtttcaattcaa tggatttgg 29 43 29 DNA Artificial Sequence #3R primer 43ccgctcgaga ttggtaactc tatgacatt 29 44 29 DNA Artificial Sequence #4Fprimer 44 ccgctcgaga tttctgccta tactcttaa 29 45 29 DNA ArtificialSequence #4R primer 45 ccgctcgagt agtaaacaaa cgaatacct 29 46 29 DNAArtificial Sequence #5F primer 46 ccgctcgagt tggggctaag aatggactt 29 4729 DNA Artificial Sequence #5R primer 47 ccgctcgaga caacatgaat gactgatct29 48 29 DNA Artificial Sequence #6F primer 48 ccgctcgagg ggtgtgcctggaaagttca 29 49 29 DNA Artificial Sequence #6R primer 49 ccgctcgagcaaagaaaaag aaaagggaa 29 50 29 DNA Artificial Sequence #7F primer 50ccgctcgagc aaaactttag gaataccgg 29 51 29 DNA Artificial Sequence #7Rprimer 51 ccgctcgagg aagaaaaaaa ccttaattt 29 52 29 DNA ArtificialSequence #8F primer 52 ccgctcgagc gaatgtgaat gcgctaatc 29 53 29 DNAArtificial Sequence #8R primer 53 ccgctcgagg ggctggcgta ggcacacaa 29 5429 DNA Artificial Sequence #9F primer 54 ccgctcgaga gacaaaaaat aggaaaagt29 55 29 DNA Artificial Sequence #9R primer 55 ccgctcgagg tagcaggtttcatgtggtg 29 56 29 DNA Artificial Sequence #10F primer 56 ccgctcgagttagtgcggaa tactttcct 29 57 29 DNA Artificial Sequence #10R primer 57ccgctcgagt gagaaccatc actaattaa 29 58 29 DNA Artificial Sequence #11Fprimer 58 ccgctcgagc agtgctttca acacctttt 29 59 29 DNA ArtificialSequence #11R primer 59 ccgctcgagg ctcggctctc gatgagaat 29 60 29 DNAArtificial Sequence #12F primer 60 ccgctcgagg tagctgaaaa gtccatcta 29 6129 DNA Artificial Sequence #12R primer 61 ccgctcgagt ctgacatggcacacagatg 29 62 29 DNA Artificial Sequence #13F primer 62 ccgctcgaggagattatctt gggatctat 29 63 29 DNA Artificial Sequence #13R primer 63ccgctcgagt agtatcattc ataattcga 29 64 29 DNA Artificial Sequence #14Fprimer 64 ccgctcgagg ttttggctca atgggaccg 29 65 29 DNA ArtificialSequence #14R primer 65 ccgctcgaga ttagccgtaa atttgtgaa 29 66 29 DNAArtificial Sequence #15F primer 66 ccgctcgagc tccacatacc aatcactcg 29 6729 DNA Artificial Sequence #15R primer 67 ccgctcgagt agttaaacgttttattagc 29

What is claimed is:
 1. A method of isolating a fungal invasin gene, saidmethod comprising (a) providing a fungus expressing a gene operablylinked to a fungal invasin gene promoter; (b) mutagenizing said fungus;(c) measuring expression of the gene, wherein an increase or decrease inthe expression of said gene identifies a mutation in said invasin gene;and (d) using said mutation as a marker for isolating said invasin gene.2. The method of claim 1, wherein said fungus is a wild-type strain. 3.The method of claim 2, wherein said wild-type strain is Saccharomycescerevisiae.
 4. The method of claim 1, wherein said fungus is a mutantstrain.
 5. The method of claim 1, wherein said gene comprises a fungalinvasin gene.
 6. The method of claim 5, wherein said fungal invasin geneis FLO11 or MUC1.
 7. The method of claim 5, wherein said fungal invasingene is AFL1, DHH1, INV1, INV5, INV6, INV7, INV8, INV9, INV10, INV11,INV12, INV13, INV14, INV15, BEM2, CDC25, HOG1, IRA1, MCM1, MGA1, PET9,PHD2, PHO23, RIM1, PTC1, RIM15, SFL1, SRB11, SSD1, STE21, STP22, SWI4,TPK2, TPK3, or YPR1.
 8. The method of claim 1, wherein said genecomprises a reporter gene.
 9. The method of claim 8, wherein saidreporter gene is lacZ, URA3, or HIS3.
 10. The method of claim 1, whereinsaid fungal invasin gene promoter is the FLO11, MUC1, STA1, STA2, orSTA3 gene promoter.
 11. The method of claim 1, wherein said fungalinvasin gene promoter is the AFL1, DHH1, INV1, INV5, INV6, INV7, INV8,INV9, INV10, INV11, INV12, INV13, INV14, INV15, BEM2, CDC25, RIM1, HOG1,IRA1, MCM1, MGA1, PET9, PHD2, PHO23, PTC1, RIM15, SFL1, SRB11, SSD1,STE21, STP22, SWI4, TPK2, TPK3, or YPR1 gene promoter.
 12. The method ofclaim 1, wherein said fungal invasin gene promoter is a fragment ordeletion of the FLO11, MUC1, STA1, STA2, or STA3 gene promoter.
 13. Themethod of claim 12, wherein said fragment is fused to a basal promoter.14. The method of claim 13, wherein said basal promoter is PGK1, ADH1,GAL1-10, tet-R, MET25, CYC1 or CUP1.
 15. The method of claim 1, whereinsaid gene expression is measured by assaying the protein level of theexpressed gene.
 16. The method of claim 1, wherein said gene expressionis measured by assaying the RNA level of the expressed gene.